Glycosaminoglycans

ABSTRACT

Heparan sulphate HS/BMP2 is disclosed, together with the use of HS/BMP2 in the repair and regeneration of bone tissue.

RELATED APPLICATION

This application is a continuation-in-part application of U.S.application Ser. No. 13/062,364, filed Mar. 4, 2011 entitled “HeparanSulphate Which Binds BMP2.”U.S. application Ser. No. 13/062,364, is a 35U.S.C. §371 national phase application of PCT/SG2009/000328, filed Sep.11, 2009 (WO 2010/030244), entitled “A Heparan Sulphate Which BindsBMP2.” PCT/SG2009/000328 claims priority to U.S. Provisional ApplicationSer. No. 61/096,274, filed Sep. 11, 2008 and United Kingdom ApplicationSerial No. 0818255.2, filed Oct. 6, 2008. Each of these applications isincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to glycosaminoglycans capable of bindingto BMP2, including their isolation and identification, and the use ofthe isolated glycosaminoglycans in the growth and/or development oftissue.

Incorporated by reference herein in its entirety is the Sequence Listingentitled “Sequence_Listing.txt”, created Sep. 5, 2012, size of 6kilobytes.

BACKGROUND TO THE INVENTION

Glycosaminoglycans (GAGs) are complex carbohydrate macromoleculesresponsible for performing and regulating a vast number of essentialcellular functions.

GAGs have been implicated in the modulation or mediation of manysignalling systems in concert with the many hundreds of knownheparin-binding growth and adhesive factors. It is contemplated that theassociation of growth factors with GAGs modulates their variousactivities with a diverse range of actions, such as lengthening theirhalf-lives by protecting them from proteolytic degradation, modulatinglocalisation of these cytokines at the cell surface, mediating molecularinteractions and stabilising ligand-receptor complexes.

There are an ever increasing number of identified heparin-binding growthfactors, adding to the hundreds already known, most of which werepurified by heparin affinity chromatography. They include the largefibroblast growth factor (FGF) family, the PDGFs, the pleiotropinsthrough to the TGF-β superfamily of cytokines. This latter family offactors encompasses the osteo-inductive bone morphogenetic protein (BMP)subfamily, so named for their ability to induce ectopic bone formation.

The nature and effect of the interaction of GAGs and growth factorsremains unclear. Although the interaction between FGF2 and particularsaccharide sequences found within heparin has been shown to be of highaffinity, it remains generally unclear whether the association betweenother growth factors and heparans involves a high affinity or specificbinding interaction between an amino acid sequence or conformationalepitope on the protein growth factor and a saccharide sequence embeddedin the GAG, or whether the association is mediated by lower affinity,non-specific interactions between the GAG and protein growth factor.

If interactions between GAGs and proteins resident in, or secreted into,the extracellular matrix are specific, the binding partners need to beidentified in order to unravel the interactions and understand how theseinteractions may be used or modulated to provide new treatments.

A major question that arises is, therefore, whether there are saccharidesequences embedded in the chains of GAG molecules that match primaryamino acid sequence/3-dimensional tertiary conformation within thepolypeptide backbone of growth factors so controlling their association,and thus bioactivity, with absolute, or at least relative, specificity.

Bone morphogenetic protein 2 (also called bone morphogenic protein 2,BMP2 or BMP-2) is a member of the TGF-β superfamily strongly implicatedin the development of bone and cartilage. It is an osteogenic protein,i.e. is a potent inducer of osteoblast differentiation (Marie et al.(2002) Regulation of human cranial osteoblast phenotype by FGF-2, FGFR-2and BMP-2 signaling”. Histol. Histopathol. 17 (3): 877-85). Implantationof collagen sponges impregnated with BMP2 has been shown to induce newbone formation (Geiger M, Li R H, Friess W (November 2003). Collagensponges for bone regeneration with rhBMP-2. Adv. Drug Deliv. Rev. 55(12): 1613-29.). Recombinant human BMP2 is available for orthopaedic usein USA (e.g. INFUSE® Bone Graft, Medtronic Inc, USA).

SUMMARY OF THE INVENTION

The present invention concerns a heparan sulphate preparation, heparansulphate HS/BMP2. HS/BMP2 has been found to enhance the generation,repair and regeneration of connective tissue.

In one aspect of the present invention heparan sulphate HS/BMP2 isprovided. HS/BMP2 may be provided in isolated form or in substantiallypurified form. This may comprise providing a composition in which theheparan sulphate component is at least 80% HS/BMP2, more preferably oneof at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%.

In preferred embodiments, HS/BMP2 is capable of binding a peptide orpolypeptide having, or consisting of, the amino acid sequence of SEQ IDNO:1 or 6. In some embodiments this peptide is SEQ ID NO:1 or 6, inother embodiments it is a BMP2 protein. In some embodiments HS/BMP2binds to a peptide having or consisting of the amino acid sequence ofSEQ ID NO:1 or 6 with a K_(D) of less than 100 μM, more preferably lessthan one of 50 μM, 40 μM, 30 μM, 20 μM, or 10 μM.

In some preferred embodiments HS/BMP2 is N-sulfated. This may compriseN-sulfation of N-acetyl-D-glucosamine (GlcNAc) residues in the heparansulphate oligosaccharide chain. Preferably at least 80% ofN-acetyl-D-glucosamine (GlcNAc) residues in the HS/BMP2 are N-sulfated.In some embodiments this may be one of at least 85%, 90%, 95%, 96%, 97%,98%, 99% or 100%.

In some preferred embodiments HS/BMP2 is 6-O-sulfated (O-sulphation atC6 of N-sulphoglucosamine (GlcNS) residues). Preferably at least 80% ofN-sulphoglucosamine (GlcNS) residues in the HS/BMP2 are 6-O-sulfated. Insome embodiments this may be one of at least 85%, 90%, 95%, 96%, 97%,98%, 99% or 100%.

HS/BMP2 may be obtained, identified, isolated or enriched according tothe inventors' methodology described herein, which may comprise thefollowing steps:

-   -   (i) providing a solid support having polypeptide molecules        adhered to the support, wherein the polypeptide comprises a        heparin-binding domain having the amino acid sequence of SEQ ID        NO:1 or 6;    -   (ii) contacting the polypeptide molecules with a mixture        comprising glycosaminoglycans such that        polypeptide-glycosaminoglycan complexes are allowed to form;    -   (iii) partitioning polypeptide-glycosaminoglycan complexes from        the remainder of the mixture;    -   (iv) dissociating glycosaminoglycans from the        polypeptide-glycosaminoglycan complexes;    -   (v) collecting the dissociated glycosaminoglycans.

In the inventors' methodology the mixture may compriseglycosaminoglycans obtained from commercially available sources. Onesuitable source is an heparan sulphate fraction, e.g. a commerciallyavailable heparan sulphate. One suitable heparan sulphate fraction canbe obtained during isolation of heparin from pig intestinal mucosa (e.g.Celsus Laboratories Inc.—sometimes called “Celsus HS”). Other suitablesources of heparan sulphate include heparan sulphate from any mammal(human or non-human), particularly from the kidney, lung or intestinalmucosa. In some embodiments the heparan sulphate is from pig (porcine)or cow (bovine) intestinal mucosa, kidney or lung. Another suitablesource is osteoblast extracellular matrix material, or a heparansulphate fraction obtained from osteoblast extracellular matrixmaterial.

In another aspect of the present invention a composition comprisingHS/BMP2 according to any one of the aspects above and BMP2 protein isprovided.

In one aspect of the present invention a pharmaceutical composition ormedicament is provided comprising HS/BMP2 in accordance with the aspectsdescribed above. The pharmaceutical composition or medicament mayfurther comprise a pharmaceutically acceptable carrier, adjuvant ordiluent.

In some embodiments the pharmaceutical composition is for use in amethod of treatment, the method comprising the repair and/orregeneration of a broken bone. In some embodiments the pharmaceuticalcomposition or medicament may further comprise BMP2 protein. In someembodiments the pharmaceutical composition or medicament may furthercomprise mesenchymal stem cells.

In another aspect of the present invention HS/BMP2 is provided for usein a method of medical treatment. The method of medical treatment maycomprise a method of wound healing in vivo, the repair and/orregeneration of connective tissue, the repair and/or regeneration ofbone and/or the repair and/or regeneration of bone in a mammal or ahuman.

In a related aspect of the present invention the use of HS/BMP2 in themanufacture of a medicament for use in a method of medical treatment isprovided. In some embodiments the method of medical treatment comprisesthe repair and/or regeneration of a broken bone in a mammal or a human.

In a further aspect of the present invention a biocompatible implant orprosthesis comprising a biomaterial and HS/BMP2 is provided. In someembodiments the implant or prosthesis is coated with HS/BMP2. In someembodiments the implant or prosthesis is impregnated with HS/BMP2. Theimplant or prosthesis may be further coated or impregnated with BMP2protein and/or with mesenchymal stem cells.

In another aspect of the present invention a method of forming abiocompatible implant or prosthesis is provided, the method comprisingthe step of coating or impregnating a biomaterial with HS/BMP2. In someembodiments the method further comprises coating or impregnating thebiomaterial with one or both of BMP2 protein and mesenchymal stem cells.

In another aspect of the present invention a method of treating a bonefracture in a patient is provided, the method comprising administrationof a therapeutically effective amount of HS/BMP2 to the patient. In someembodiments the method comprises administering HS/BMP2 to the tissuesurrounding the fracture. In some embodiments the method comprisesinjection of HS/BMP2 to the tissue surrounding the fracture. In suchmethods the HS/BMP2 may be formulated as a pharmaceutical composition ormedicament comprising HS/BMP2 and a pharmaceutically acceptable carrier,adjuvant or diluent.

In some embodiments the method may further comprise administering BMP2protein to the patient. In such methods the HS/BMP2 and BMP2 protein maybe formulated as a pharmaceutical composition comprising HS/BMP2 andBMP2 protein and a pharmaceutically acceptable carrier, adjuvant ordiluent.

In some embodiments the method may further comprise administeringmesenchymal stem cells to the patient. In such methods at least two ofHS/BMP2, BMP2 protein and mesenchymal stem cells may be formulated in apharmaceutical composition comprising at least two of the HS/BMP2, BMP2protein and mesenchymal stem cells and a pharmaceutically acceptablecarrier, adjuvant or diluent.

Preferably, the HS/BMP2, BMP2 protein and mesenchymal stem cells arerespectively provided in therapeutically effective amounts. In someembodiments the method of treating bone fracture further comprises thestep of formulating therapeutically effective amounts of HS/BMP2, and/orBMP2 protein and/or mesenchymal stem cells as a pharmaceuticalcomposition comprising the HS/BMP2, and/or BMP2 protein and/ormesenchymal stem cells and a pharmaceutically acceptable carrier,adjuvant or diluent, wherein the pharmaceutical composition isadministered to the patient.

In another aspect of the present invention a method of treating a bonefracture in a patient is provided, the method comprising surgicallyimplanting a biocompatible implant or prosthesis, which implant orprosthesis comprises a biomaterial and HS/BMP2, into tissue of thepatient at or surrounding the site of fracture.

In some embodiments the implant or prosthesis is coated with HS/BMP2. Insome embodiments the implant or prosthesis is impregnated with HS/BMP2.In some embodiments the implant or prosthesis is further impregnatedwith one or both of BMP2 protein and mesenchymal stem cells.

In a further aspect of the present invention culture media is provided,the culture media comprising HS/BMP2.

In another aspect of the present invention the use of HS/BMP2 in cellculture in vitro is provided. In a related aspect of the presentinvention the use of HS/BMP2 in the growth of connective tissue in vitrois provided. In another related aspect of the present invention a methodfor growing connective tissue in vitro is provided, the methodcomprising culturing mesenchymal stem cells in contact with exogenouslyadded HS/BMP2.

In yet a further aspect of the present invention a method of promotingosteogenesis is provided, the method comprising administering HS/BMP2 tobone precursor cells or bone stem cells. The method may involvecontacting the bone precursor cells or bone stem cells with HS/BMP2 invitro or in vivo. In some embodiments the bone precursor cells or bonestem cells are contacted with BMP2 protein simultaneously with HS/BMP2.In preferred embodiments the bone precursor or bone stem cells aremesenchymal stem cells.

In yet a further aspect of the present invention a method for therepair, replacement or regeneration of bone tissue in a human or animalpatient in need of such treatment is provided, the method comprising:

-   -   (i) culturing mesenchymal stem cells in vitro in contact with        HS/BMP2 for a period of time sufficient for said cells to form        bone tissue;    -   (ii) collecting said bone tissue;    -   (iii) implanting said bone tissue into the body of the patient        at a site of injury or disease to repair, replace or regenerate        bone tissue in the patient.

In some embodiments the method further comprises contacting themesenchymal stem cells in culture with exogenous BMP2 protein.

In another aspect of the present invention bone tissue obtained by invitro culture of mesenchymal stem cells in the presence of HS/BMP2 isprovided. In some embodiments the bone tissue is obtained by in vitroculture of mesenchymal stem cells in the presence of HS/BMP2 and BMP2protein.

In a further aspect of the present invention a method of culturingmesenchymal stem cells is provided, the method comprising culturingmesenchymal stem cells in contact with HS/BMP2.

In yet a further aspect of the present invention a kit of parts isprovided, the kit comprising a predetermined amount of HS/BMP2 and apredetermined amount of BMP2. The kit may comprise a first containercontaining the predetermined amount of HS/BMP2 and a second containercontaining the predetermined amount of BMP2. The kit may furthercomprise a predetermined amount of mesenchymal stem cells. The kit maybe provided for use in a method of medical treatment. The method ofmedical treatment may comprise a method of wound healing in vivo, therepair and/or regeneration of connective tissue, the repair and/orregeneration of bone and/or the repair and/or regeneration of bone in amammal or a human. The kit may be provided together with instructionsfor the administration of the HS/BMP2, BMP2 protein and/or mesenchymalstem cells separately, sequentially or simultaneously in order toprovide the medical treatment.

In a further aspect of the present invention products are provided, theproducts containing therapeutically effective amounts of:

-   -   (i) HS/BMP2; and one or both of    -   (ii) BMP2 protein;    -   (iii) Mesenchymal stem cells,        for simultaneous, separate or sequential use in a method of        medical treatment. The method of medical treatment may comprise        a method of wound healing in vivo, the repair and/or        regeneration of connective tissue, the repair and/or        regeneration of bone and/or the repair and/or regeneration of        bone in a mammal or a human. The products may optionally be        formulated as a combined preparation for coadministration.

Further aspects of the present invention are set out below.

In one aspect of the present invention a GAG is provided having highbinding affinity for BMP2. More preferably the GAG is a heparan sulphate(HS). In one embodiments the HS was isolated from a GAG mixture obtainedfrom the extracellular matrix of osteoblasts by following themethodology described herein in which a polypeptide comprising theheparin-binding domain of BMP2 (SEQ ID NO:1) was attached to a solidsupport and GAG-polypeptide complexes were allowed to form. Dissociationof the GAG component from the GAG-polypeptide complexes led to isolationof a unique HS herein called “HS/BMP2” (sometimes called “HS-3” or“HS3”).

In another embodiment the same methodology was used to isolate HS/BMP2from a commercially available heparan sulphate (Celsus HS) obtainedduring isolation of heparin from pig intestinal mucosa and availablefrom Celsus Laboratories Inc, Cincinnatti, USA (e.g. INW-08-045, HeparanSulphate I, Celsus Lab Inc, HO-03102, HO-10595, 10×100 mg).

It is the inventors belief that HS/BMP2 can be obtained from HSfractions obtained from a plurality of sources, including mammalian(human and non-human) tissue and/or extracellular matrix.

Accordingly, in one aspect of the present invention HS/BMP2 is provided.HS/BMP2 may be provided in isolated or purified form. In another aspectculture media comprising HS/BMP2 is provided.

In yet another aspect of the present invention a pharmaceuticalcomposition or medicament comprising HS/BMP2 is provided, optionally incombination with a pharmaceutically acceptable carrier, adjuvant ordiluent. In some embodiments pharmaceutical compositions or medicamentsmay further comprise BMP2 protein. Pharmaceutical compositions ormedicaments comprising HS/BMP2 are provided for use in the prevention ortreatment of injury or disease. The use of HS/BMP2 in the manufacture ofa medicament for the prevention or treatment of injury or disease isalso provided.

In a further aspect of the present invention, a method of preventing ortreating injury or disease in a patient in need of treatment thereof isprovided, the method comprising administering an effective amount ofHS/BMP2 to the patient. The administered HS/BMP2 may be formulated in asuitable pharmaceutical composition or medicament and may furthercomprise a pharmaceutically acceptable carrier, adjuvant or diluent.Optionally, the pharmaceutical composition or medicament may alsocomprise BMP2 protein.

In another aspect of the present invention a method of promoting orinhibiting osteogenesis (the formation of bone cells and/or bone tissue)is provided comprising administering HS/BMP2 to bone precursor cells orbone stem cells.

In another aspect of the present invention a method of promoting orinhibiting the formation of cartilage tissue (chondrogenesis) isprovided, comprising administering HS/BMP2 to cartilage precursor cellsor cartilage stem cells.

The methods of stimulating or inhibiting osteogenesis or formation ofcartilage tissue may be conducted in vitro by contacting bone orcartilage precursor or stem cells with HS/BMP2, optionally in thepresence of exogenously added BMP2 protein. The precursor cells or stemcells may be mesenchymal stem cells. Where tissue formation is promoted,the tissue formed may be collected and used for implantation into ahuman or animal patient.

Accordingly, in one aspect of the present invention, connective tissueis provided wherein the connective tissue is obtained by in vitroculture of mesenchymal stem cells in the presence of HS/BMP2 (i.e.exogenous HS/BMP2), and optionally in the presence of BMP2 (i.e.exogenous BMP2). The connective tissue may be bone, cartilage, muscle,fat, ligament or tendon.

The prevention or treatment of disease using HS/BMP2 may involve therepair, regeneration or replacement of tissue, particularly connectivetissue such as bone, cartilage, muscle, fat, ligament or tendon.

In patients having a deterioration of one of these tissues,administration of HS/BMP2 to the site of deterioration may be used tostimulate the growth, proliferation and/or differentiation of tissue atthat site. For example, stimulation of mesenchymal stem cells presentat, or near to, the site of administration may lead, preferably whenBMP2 is also present at the site, to the proliferation anddifferentiation of the mesenchymal stem cells into the appropriateconnective tissue, thereby providing for replacement/regeneration of thedamaged tissue and treatment of the injury.

Alternatively, connective tissue obtained from in vitro culture ofmesenchymal stem cells in contact with HS/BMP2 may be collected andimplanted at the site of injury or disease to replace damaged ordeteriorated tissue. The damaged or deteriorated tissue may optionallyfirst be excised from the site of injury or disease.

In another aspect, a pharmaceutical composition may be providedcontaining stem cells, preferably mesenchymal stem cells, and HS/BMP2.Administration, e.g. injection, of the composition at the site ofinjury, disease or deterioration provides for the regeneration of tissueat the site.

Accordingly, HS/BMP2 is useful in wound healing in vivo, includingtissue repair, regeneration and/or replacement (e.g. healing of scartissue or a broken bone) effected by direct application of HS/BMP2,optionally in combination with BMP2 and/or stem cells, to the patientrequiring treatment. HS/BMP2 is also useful in the in vitro generationof tissue suitable for implantation into a patient in need of tissuerepair, regeneration and/or replacement.

The following numbered paragraphs (paras.) contain statements of broadcombinations of the inventive technical features herein disclosed:—

1. A method of isolating glycosaminoglycans capable of binding to aprotein having a heparin-binding domain, the method comprising:

-   -   (i) providing a solid support having polypeptide molecules        adhered to the support, wherein the polypeptide comprises a        heparin-binding domain;    -   (ii) contacting the polypeptide molecules with a mixture        comprising glycosaminoglycans such that        polypeptide-glycosaminoglycan complexes are allowed to form;    -   (iii) partitioning polypeptide-glycosaminoglycan complexes from        the remainder of the mixture;    -   (iv) dissociating glycosaminoglycans from the        polypeptide-glycosaminoglycan complexes;    -   (v) collecting the dissociated glycosaminoglycans.        2. A method of identifying glycosaminoglycans capable of        stimulating or inhibiting the growth and/or differentiation of        cells and/or tissues, the method comprising:    -   (i) providing a solid support having polypeptide molecules        adhered to the support, wherein the polypeptide comprises a        heparin-binding domain;    -   (ii) contacting the polypeptide molecules with a mixture        comprising glycosaminoglycans such that        polypeptide-glycosaminoglycan complexes are allowed to form;    -   (iii) partitioning polypeptide-glycosaminoglycan complexes from        the remainder of the mixture;    -   (iv) dissociating glycosaminoglycans from the        polypeptide-glycosaminoglycan complexes;    -   (v) collecting the dissociated glycosaminoglycans;    -   (vi) adding the collected glycosaminoglycans to cells or tissues        in which a protein containing the amino acid sequence of the        heparin-binding domain is present;    -   (vii) measuring one or more of: proliferation of the cells,        differentiation of the cells, expression of one or more        protein/glycoprotein/glycolipid markers markers.        3. The method of paragraph 1 or 2 wherein the mixture comprising        glycosaminoglycans contains extracellular matrix material.        4. The method of paragraph 3 wherein the extracellular matrix        material is derived from connective tissue or connective tissue        cells.        5. The method of any one of paragraphs 1 to 4 wherein the        mixture comprising glycosaminoglycans contains one or more of a        dextran sulphate, a chondroitin sulphate, a heparan sulphate.        6. The method of any one of paragraphs 1 to 5 wherein the        mixture comprising glycosaminoglycans has been enriched for one        of dextran sulphate, chondroitin sulphate, heparan sulphate.        7. The method of any one of paragraphs 1 to 6 wherein the method        further comprises subjecting the collected glycosaminoglycans to        further analysis in order to determine structural        characteristics of the GAG.        8. The method of any one of paragraphs 1 to 7 wherein the        glycosaminoglycan-polypeptide complexes are contacted with a        lyase.        9. The method of any one of paragraphs 1 to 8 wherein the        polypeptide is, or comprises, one of SEQ ID NO.s: 1 or 2.        10. Heparan sulphate HS/BMP2.        11. Culture media comprising HS/BMP2.        12. A pharmaceutical composition or medicament comprising        HS/BMP2.        13. The pharmaceutical composition or medicament of paragraph 12        further comprising a pharmaceutically acceptable carrier,        adjuvant or diluent.        14. The pharmaceutical composition or medicament of paragraph 12        or 13 further comprising BMP2 protein.        15. A pharmaceutical compositions or medicament according to any        one of paragraphs 12 to 14 for use in the prevention or        treatment of injury or disease.        16. Use of HS/BMP2 in the manufacture of a medicament for the        prevention or treatment of injury or disease.        17. The pharmaceutical composition or use of paragraph 15 or 16        wherein the prevention or treatment is chosen from: repair,        regeneration or replacement of connective tissue and wound        healing.        18. A method of preventing or treating injury or disease in a        patient in need of treatment thereof, the method comprising        administering an effective amount of HS/BMP2 to the patient.        19. The method of paragraph 18 wherein the administered HS/BMP2        is formulated as a pharmaceutical composition or medicament.        20. The method of paragraph 19 wherein the pharmaceutical        composition or medicament further comprises BMP2 protein.        21. A method of promoting or inhibiting osteogenesis comprising        administering HS/BMP2 to bone precursor cells or bone stem        cells.        22. The method of paragraph 21 wherein the bone precursor cells        or bone stem cells are contacted with HS/BMP2 in vitro.        23. The method of paragraph 21 wherein the bone precursor cells        or bone stem cells are contacted with HS/BMP2 in vivo.        24. The method of any one of paragraphs 21 to 23 wherein the        bone precursor cells or bone stem cells are contacted with BMP2        simultaneously with HS/BMP2.        25. A method of promoting or inhibiting the formation of        cartilage tissue comprising administering HS/BMP2 to cartilage        precursor cells or cartilage stem cells.        26. The method of paragraph 25 wherein the cartilage precursor        cells or cartilage stem cells are contacted with HS/BMP2 in        vitro.        27. The method of paragraph 25 wherein the cartilage precursor        cells or cartilage stem cells are contacted with HS/BMP2 in        vivo.        28. The method of any one of paragraphs 25 to 27 wherein the        cartilage precursor cells or cartilage stem cells are contacted        with BMP2 simultaneously with HS/BMP2.        29. A method for the repair, replacement or regeneration of        connective tissue in a human or animal patient in need of such        treatment, the method comprising:    -   (i) culturing mesenchymal stem cells in vitro in contact with        HS/BMP2 for a period of time sufficient for said cells to form        connective tissue;    -   (ii) collecting said connective tissue;    -   (iii) implanting said connective tissue into the body of the        patient at a site of injury or disease to repair, replace or        regenerate connective tissue in the patient.        30. The method of paragraph 29 further comprising contacting the        mesenchymal cells in culture with exogenous BMP2.        31. Connective tissue obtained by in vitro culture of        mesenchymal stem cells in the presence of HS/BMP2.        32. Connective tissue as paragraphed in paragraph 31, wherein        the mesenchymal cells are cultured in the presence of exogenous        BMP2, and optionally in the presence of BMP2.        33. A pharmaceutical composition comprising stem cells and        HS/BMP2.        34. The pharmaceutical composition of paragraph 32 wherein the        stem cells are mesenchymal stem cells.        35. The pharmaceutical composition of paragraphs 33 or 34        wherein the composition further comprises BMP2.        36. A pharmaceutical composition according to any of paragraphs        33 to 35 for use in the treatment of injury or disease.        37. A method for the treatment of injury or disease in a patient        in need of treatment thereof comprising administering to the        patient a pharmaceutical composition comprising stem cells and        HS/BMP2.        38. The method of paragraph 37 wherein the stem cells are        mesenchymal stem cells.        39. The method of paragraph 37 or 38 wherein the method further        comprises administering BMP2 to the patient.        40. Use of HS/BMP2 in the growth of connective tissue in vitro.        41. A method for growing connective tissue in vitro comprising        culturing mesenchymal stem cells in contact with exogenously        added HS/BMP2.        42. A biological implant comprising a solid or semi-solid matrix        material impregnated with HS/BMP2.        43. The biological implant of paragraph 42 further impregnated        with BMP2.        44. The biological implant of paragraph 42 or 43 further        impregnated with mesenchymal stem cells.        45. A kit comprising a predetermined amount of a        glycosaminoglycan having high affinity for a protein having a        heparin-binding domain and a predetermined amount of said        protein.        46. The kit of paragraph 45 wherein the glycosaminoglycan is        HS/BMP2 and the protein is BMP2.        The following numbered paragraphs contain further statements of        broad combinations of the inventive technical features herein        disclosed:—        1. Heparan sulphate HS/BMP2.        2. HS/BMP2 in isolated or substantially purified form.        3. HS/BMP2 according to paragraph 1 or 2 wherein the HS/BMP2 is        capable of binding SEQ ID NO:1 or 6.        4. HS/BMP2 according to paragraph 3 which binds to SEQ ID NO:1        with a K_(D) of less than 100 μM.        5. HS/BMP2 according to any one of paragraphs 1 to 4 wherein the        HS/BMP2 is N-sulfated.        6. HS/BMP2 according to any one of paragraphs 1 to 5 wherein the        HS/BMP2 is 6-O-sulfated.        7. HS/BMP2 or HS/BMP2 in isolated or substantially purified form        according to any one of paragraphs 1 to 6 obtained by a method        comprising:    -   (i) providing a solid support having polypeptide molecules        adhered to the support, wherein the polypeptide comprises a        heparin-binding domain having the amino acid sequence of SEQ ID        NO:1 or 6;    -   (ii) contacting the polypeptide molecules with a mixture        comprising glycosaminoglycans such that        polypeptide-glycosaminoglycan complexes are allowed to form;    -   (iii) partitioning polypeptide-glycosaminoglycan complexes from        the remainder of the mixture;    -   (iv) dissociating glycosaminoglycans from the        polypeptide-glycosaminoglycan complexes;    -   (v) collecting the dissociated glycosaminoglycans.        8. The method of paragraph 7 wherein the mixture comprising        glycosaminoglycans is obtained from osteoblast extracellular        matrix material.        9. A composition comprising HS/BMP2 according to any one of        paragraphs 1 to 8 and BMP2 protein.        10. A pharmaceutical composition or medicament comprising        HS/BMP2 according to any one of paragraphs 1 to 8.        11. The pharmaceutical composition or medicament of paragraph 10        for use in a method of treatment, the method comprising the        repair and/or regeneration of a broken bone.        12. The pharmaceutical composition or medicament of paragraph 10        or 11 further comprising BMP2 protein.        13. The pharmaceutical composition or medicament of any one of        paragraphs 10 to 12 wherein the pharmaceutical composition or        medicament further comprises mesenchymal stem cells.        14. HS/BMP2 according to any one of paragraphs 1 to 8 for use in        a method of medical treatment.        15. HS/BMP2 for use in a method of medical treatment according        to paragraph 14 or 15 wherein the method of medical treatment        comprises a method of wound healing in vivo.        16. HS/BMP2 for use in a method of medical treatment according        to paragraph 14 or 15 wherein the method of medical treatment        comprises repair and/or regeneration of connective tissue.        17. HS/BMP2 for use in a method of medical treatment according        to paragraph 14 or 15 wherein the method of medical treatment        comprises repair and/or regeneration of bone.        18. HS/BMP2 for use in a method of medical treatment according        to paragraph 14 wherein the method of medical treatment        comprises repair and/or regeneration of bone in a mammal or a        human.        19. Use of HS/BMP2 according to any one of paragraphs 1 to 8 in        the manufacture of a medicament for the repair and/or        regeneration of a broken bone in a mammal or a human.        20. A biocompatible implant or prosthesis comprising a        biomaterial and HS/BMP2.        21. The implant or prosthesis of paragraph 20 wherein the        implant or prosthesis is coated with HS/BMP2.        22. The implant or prosthesis of paragraph 20 wherein the        implant or prosthesis is impregnated with HS/BMP2.        23. The implant or prosthesis of any one of paragraphs 20 to 22        wherein the implant or prosthesis is further coated or        impregnated with BMP2 protein and/or with mesenchymal stem        cells.        24. A method of forming a biocompatible implant or prosthesis,        the method comprising the step of coating or impregnating a        biomaterial with HS/BMP2.        25. The method of paragraph 24 wherein the method further        comprises coating or impregnating the biomaterial with one or        both of BMP2 protein and mesenchymal stem cells.        26. A method of treating a bone fracture in a patient, the        method comprising administration of a therapeutically effective        amount of HS/BMP2 to the patient.        27. The method of paragraph 26 wherein the method comprises        administering HS/BMP2 to the tissue surrounding the fracture.        28. The method of paragraph 26 or 27 wherein administration of        HS/BMP2 comprises injection of HS/BMP2 to the tissue surrounding        the fracture.        29. The method of any one of paragraphs 26 to 28 wherein the        HS/BMP2 is formulated as a pharmaceutical composition or        medicament comprising HS/BMP2 and a pharmaceutically acceptable        carrier, adjuvant or diluent.        30. The method of any one of paragraphs 26 to 29 wherein the        method further comprises administering BMP2 protein to the        patient.        31. The method of paragraph 30 wherein the HS/BMP2 and BMP2        protein are formulated as a pharmaceutical composition        comprising HS/BMP2 and BMP2 protein and a pharmaceutically        acceptable carrier, adjuvant or diluent.        32. The method of any one of paragraphs 26 to 31 wherein the        method further comprises administering mesenchymal stem cells to        the patient.        33. The method of paragraph 32 wherein at least two of the        HS/BMP2, BMP2 protein and mesenchymal stem cells are formulated        in a pharmaceutical composition comprising at least two of the        HS/BMP2, BMP2 protein and mesenchymal stem cells and a        pharmaceutically acceptable carrier, adjuvant or diluent.        34. A method of treating a bone fracture in a patient, the        method comprising surgically implanting a biocompatible implant        or prosthesis, which implant or prosthesis comprises a        biomaterial and HS/BMP2, into tissue of the patient at or        surrounding the site of fracture.        35. The method of paragraph 34 wherein the implant or prosthesis        is coated with HS/BMP2.        36. The method of paragraph 34 wherein the implant or prosthesis        is impregnated with HS/BMP2.        37. The method of any one of paragraphs 34 to 36 wherein the        implant or prosthesis is further impregnated with one or both of        BMP2 protein and mesenchymal stem cells.        38. Culture media comprising HS/BMP2.        39. Use of HS/BMP2 in cell culture in vitro.        40. Use of HS/BMP2 in the growth of connective tissue in vitro.        41. A method for growing connective tissue in vitro comprising        culturing mesenchymal stem cells in contact with exogenously        added HS/BMP2.        42. A method of promoting osteogenesis, the method comprising        administering HS/BMP2 to bone precursor cells or bone stem        cells.        43. The method of paragraph 42 wherein the bone precursor cells        or bone stem cells are contacted with HS/BMP2 in vitro.        44. The method of paragraph 42 wherein the bone precursor cells        or bone stem cells are contacted with HS/BMP2 in vivo.        45. The method of any one of paragraphs 42 to 44 wherein the        bone precursor cells or bone stem cells are contacted with BMP2        protein simultaneously with HS/BMP2.        46. The method of any one of paragraphs 42 to 45 wherein the        bone precursor or bone stem cells are mesenchymal stem cells.        47. A method for the repair, replacement or regeneration of bone        tissue in a human or animal patient in need of such treatment,        the method comprising:    -   (i) culturing mesenchymal stem cells in vitro in contact with        HS/BMP2 for a period of time sufficient for said cells to form        bone tissue;    -   (ii) collecting said bone tissue;    -   (iii) implanting said bone tissue into the body of the patient        at a site of injury or disease to repair, replace or regenerate        bone tissue in the patient.        48. The method of paragraph 47 further comprising contacting the        mesenchymal stem cells in culture with exogenous BMP2 protein.        49. Bone tissue obtained by in vitro culture of mesenchymal stem        cells in the presence of HS/BMP2.        50. Bone tissue as paragraphed in paragraph 49, wherein the        mesenchymal stem cells are cultured in the presence of BMP2        protein.        51. A method of culturing mesenchymal stem cells comprising        culturing mesenchymal stem cells in contact with HS/BMP2.        52. A kit comprising a predetermined amount of HS/BMP2 and a        predetermined amount of BMP2.        53. Products containing therapeutically effective amounts of:    -   (i) HS/BMP2; and one or both of    -   (ii) BMP2 protein;    -   (iii) Mesenchymal stem cells,        for simultaneous, separate or sequential use in a method of        medical treatment.

DESCRIPTION OF PREFERRED EMBODIMENTS HS/BMP2

The present invention relates to HS/BMP2, which is obtainable by methodsof enriching mixtures of compounds containing one or more GAGs that bindto a polypeptide corresponding to the heparin-binding domain of BMP2.The enrichment process may be used to isolate HS/BMP2.

The present invention also relates to mixtures of compounds enrichedwith HS/BMP2, and methods of using such mixtures.

HS/BMP2 is believed to potentiate (e.g. agonize) the activity of BMP-2and hence its ability to stimulate stem cell proliferation and boneformation.

In addition to being obtainable by the methodology described here,HS/BMP2 can also be defined functionally and structurally.

Functionally, HS/BMP2 is capable of binding a peptide having, orconsisting of, the amino acid sequence of SEQ ID NO:1 or 6. Preferably,HS/BMP2 binds the peptide of SEQ ID NO:1 or 6 with a K_(D) of less than100 μM, more preferably less than one of 50 μM, 40 μM, 30 μM, 20 μM, or10 μM.

Preferably, HS/BMP2 also binds BMP2 protein with a K_(D) of less than100 μM, more preferably less than one of 50 μM, 40 μM, 30 μM, 20 μM, or10 μM. Binding between HS/BMP2 and BMP2 protein may be determined by thefollowing assay method.

BMP2 is dissolved in Blocking Solution (0.2% gelatin in SAB) at aconcentration of 3 μg/ml and a dilution series from 0-3 μg/ml inBlocking Solution is established. Dispensing of 200 μl of each dilutionof BMP2 into triplicate wells of Heparin/GAG Binding Plates pre-coatedwith heparin; incubated for 2 hrs at 37° C., washed carefully threetimes with SAB and 200 μl of 250 ng/ml biotinylated anti-BMP2 added inBlocking Solution. Incubation for one hour at 37° C., wash carefullythree times with SAB, 200 μl of 220 ng/ml ExtrAvidin-AP added inBlocking Solution, Incubation for 30 mins at 37° C., careful washingthree times with SAB and tap to remove residual liquid, 200 μl ofDevelopment Reagent (SigmaFAST p-Nitrophenyl phosphate) added. Incubateat room temperature for 40 minutes with absorbance reading at 405 nmwithin one hour.

In this assay, binding may be determined by measuring absorbance and maybe determined relative to controls such as BMP2 protein in the absenceof added heparan sulphate, or BMP2 protein to which an heparan sulphateis added that does not bind BMP2 protein.

The binding of HS/BMP2 is preferably specific, in contrast tonon-specific binding and in the context that the HS/BMP2 can be selectedfrom other heparan sulphates and/or GAGs by a method involving selectionof heparan sulphates exhibiting a high affinity binding interaction withthe peptide of SEQ ID NO:1 or 6 or with BMP2 protein.

HS/BMP2 according to the present invention preferably enhances BMP2induced Alkaline Phoshatase (ALP) activity in cells of the mousemyoblast cell line C2C12 to a greater extent than the enhancementobtained by addition of corresponding amounts of BMP2 protein or Heparinalone. Preferably it also enhances BMP2-induced ALP activity in C2C12cells to a greater extent than that induced by combined addition ofcorresponding amounts of BMP2 protein and heparin, or of BMP2 proteinand a heparan sulphate that does not bind BMP2 protein with highaffinity (for example refer to FIG. 46).

Enhancement of ALP activity can be measured by performing the followingALP Assay. C2C12 cells are plated at 20,000 cells/cm² in a 24-well platein DMEM (e.g. Sigma-Aldrich Inc., St. Louis, Mo.) containing 10% FCS(e.g. Lonza Group Ltd., Switzerland) and antibiotics (1% Penicillin and1% Streptomycin) (e.g. Sigma-Aldrich Inc., St. Louis, Mo.) at 37° C./5%CO₂. After 24 hours, the culture media is switched to 5% FCS low serummedia containing different combinations of 100 ng/mL BMP2 (e.g. R&DSystems, Minneapolis, Minn.), 3 mg/mL Celsus HS and varyingconcentrations of BMP2-specific (+ve HS) and non-specific (−ve HS)Celsus HS preparations. Cell lysis is carried out after 3 days usingRIPA buffer containing 1% Triton X-100, 150 mM NaCl, 10 mM Tris pH 7.4,2 mM EDTA, 0.5% Igepal (NP40), 0.1% Sodium dodecyl sulphate (SDS) and 1%Protease Inhibitor Cocktail Set III (Calbiochem, Germany). The proteincontent of the cell lysate is determined by using BCA protein assay kit(Pierce Chemical Co., Rockford, Ill.). ALP activity in the cell lysateswas then determined by incubating the cell lysates withp-nitrophenylphosphate substrate (Invitrogen, Carlsbad, Calif.). Thereading is normalized to total protein amount and presented as relativeamount to the group containing BMP2 treatment alone.

Enhancement of ALP activity in C2C12 cells can also be followed byimmunohistochemical techniques, such as the following ALP stainingprotocol, illustrated by FIG. 47. ALP Staining. C2C12 cells are culturedas described in the assay methodology immediately above. After 3 days oftreatment, the cell layer is washed in PBS and stained using LeukocyteAlkaline Phosphatase Kit (e.g. Sigma-Aldrich Inc., St. Louis, Mo.)according to manufacturer's specification. The cell layer is fixed incitrate buffered 60% acetone and stained in alkaline-dye mixturecontaining Naphthol AS-MX Phosphatase Alkaline and diazonium salt.Nuclear staining is performed using Mayer's Hematoxylin solution.

These techniques can be used to identify HS/BMP2 as a heparan sulphatethat enhances a greater degree of BMP2 protein induced ALP activity inC2C12 cells compared with non-specific heparan sulphates, e.g. heparansulphates that do not bind BMP-2 protein.

HS/BMP2 according to the present invention also prolongs the effects ofBMP2 signalling to levels that equal or exceed those of heparin. Thiscan be assessed by the following assay. C2C12 cells are exposed to (i)nothing, (ii) BMP2 alone, (iii) BMP2+Heparin or (iv) BMP2+HS/BMP2 for 72hours and the phosphorylation levels of the BMP2-specific intracellularsignaling molecule Smad1/5/8 are monitored by immunoblotting.

An important functional property of HS/BMP2 is its ability to enhancethe process of bone repair, particularly in mammalian subjects. This maybe tested in a bone repair model, such as that described in Examples 8and 9, in which the speed and quality of bone repair in control animals(e.g. animals not given HS or animals given an HS that does not bindBMP2 protein or the peptide of SEQ ID NO:1 or 6) and HS/BMP2 treatedanimals is compared. Speed and quality of bone repair may be assessed byanalysing generation of bone volume at the wound site over time, e.g. byX-ray and microCT imaging analysis of the wound.

Structurally, N-sulfation of N-acetyl-D-glucosamine (GlcNAc) residues inHS/BMP2 has been found to be important as regards maintaining bindingaffinity for BMP2 protein. N-desulfation was shown to lead to asignificant reduction in BMP2 protein binding affinity (FIG. 49).

6-O-sulfation (O-sulphation at C6) of N-sulphoglucosamine (GlcNS)residues was also found to be of moderate significance as regardsmaintaining binding affinity for BMP2 protein. 6-O-desulfation led tosome reduction in BMP2 protein binding affinity (FIG. 49).

2-O-sulfation (O-sulphation at C2) of IdoA and/or D-glucuronic acid(GlcA) residues was found not to affect BMP2 protein binding. As such,HS/BMP2 may optionally be either 2-O-sulfated or 2-O-desulfated.

The disaccharide composition of HS/BMP2 is shown in FIG. 43 as measuredby lyase digestion and SAX-HPLC analysis.

The disaccharide composition of HS/BMP2 following digestion with heparinlyases I, II and III to completion and then subjecting the resultingdisaccharide fragments to capillary electrophoresis analysis is shown inFIGS. 76, 77, 78 and 79.

HS/BMP2 according to the present invention includes heparan sulphatethat has a disaccharide composition within ±10% (more preferably ±one of9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or 0.5%) of the values shown for eachdisaccharide in FIG. 43 in the column titled “BMP2-Specific HS” or inone of FIGS. 76, 77, 78 and 79 for the retained HS species (HS+ve), asdetermined respectively by lyase digestion and SAX-HPLC analysis ordigestion with heparin lyases I, II and III to completion and thensubjecting the resulting disaccharide fragments to capillaryelectrophoresis analysis.

The disaccharide composition of HS/BMP2 as determined by digestion withheparin lyases I, II and III to completion and then subjecting theresulting disaccharide fragments to capillary electrophoresis analysismay have a disaccharide composition according to any one of thefollowing:

Normalised weight Disaccharide percentage ΔHexUA,2SGlcNS,6S 14.8 ± 3.0ΔHexUA,2S-GlcNS  4.9 ± 2.0 ΔHexUA-GlcNS,6S 11.1 ± 3.0 ΔHexUA,2SGlcNAc,6S 4.8 ± 2.0 ΔHexUA-GlcNS 22.2 ± 3.0 ΔHexUA,2S-GlcNAc  1.1 ± 0.5ΔHexUA-GlcNAc,6S 10.1 ± 3.0 ΔHexUA-GlcNAc 31.1 ± 3.0 orΔHexUA,2SGlcNS,6S 14.8 ± 2.0 ΔHexUA,2S-GlcNS  4.9 ± 2.0 ΔHexUA-GlcNS,6S11.1 ± 2.0 ΔHexUA,2SGlcNAc,6S  4.8 ± 2.0 ΔHexUA-GlcNS 22.2 ± 2.0ΔHexUA,2S-GlcNAc  1.1 ± 0.5 ΔHexUA-GlcNAc,6S 10.1 ± 2.0 ΔHexUA-GlcNAc31.1 ± 2.0 or ΔHexUA,2SGlcNS,6S 14.8 ± 2.0 ΔHexUA,2S-GlcNS  4.9 ± 1.0ΔHexUA-GlcNS,6S 11.1 ± 2.0 ΔHexUA,2SGlcNAc,6S  4.8 ± 1.0 ΔHexUA-GlcNS22.2 ± 2.0 ΔHexUA,2S-GlcNAc  1.1 ± 0.5 ΔHexUA-GlcNAc,6S 10.1 ± 2.0ΔHexUA-GlcNAc 31.1 ± 3.0 or ΔHexUA,2SGlcNS,6S 14.8 ± 1.0 ΔHexUA,2S-GlcNS 4.9 ± 0.4 ΔHexUA-GlcNS,6S 11.1 ± 1.0 ΔHexUA,2SGlcNAc,6S  4.8 ± 0.6ΔHexUA-GlcNS 22.2 ± 3.0 ΔHexUA,2S-GlcNAc  1.1 ± 0.4 ΔHexUA-GlcNAc,6S10.1 ± 1.0 ΔHexUA-GlcNAc 31.1 ± 1.6 or ΔHexUA,2SGlcNS,6S  14.8 ± 0.75ΔHexUA,2S-GlcNS  4.9 ± 0.3 ΔHexUA-GlcNS,6S  11.1 ± 0.75ΔHexUA,2SGlcNAc,6S  4.8 ± 0.45 ΔHexUA-GlcNS  22.2 ± 2.25ΔHexUA,2S-GlcNAc  1.1 ± 0.3 ΔHexUA-GlcNAc,6S  10.1 ± 0.75 ΔHexUA-GlcNAc31.1 ± 1.2 or ΔHexUA,2SGlcNS,6S 14.8 ± 0.5 ΔHexUA,2S-GlcNS  4.9 ± 0.2ΔHexUA-GlcNS,6S 11.1 ± 0.5 ΔHexUA,2SGlcNAc,6S  4.8 ± 0.3 ΔHexUA-GlcNS22.2 ± 1.5 ΔHexUA,2S-GlcNAc  1.1 ± 0.2 ΔHexUA-GlcNAc,6S 10.1 ± 0.5ΔHexUA-GlcNAc 31.1 ± 0.8ororororor

Digestion of HS/BMP2 with heparin lyases I, II and III and/or capillaryelectrophoresis analysis of disaccharides is preferably performed inaccordance with Example 10.

Digestion of HS preparations with heparin lyase enzymes may be conductedas follows: HS preparations (1 mg) are each dissolved in 500 μL ofsodium acetate buffer (100 mM containing 10 mM calcium acetate, pH 7.0)and 2.5 mU each of the three enzymes is added; the samples are incubatedat 37° C. overnight (24 h) with gentle inversion (9 rpm) of the sampletubes; a further 2.5 mU each of the three enzymes is added to thesamples which are incubated at 37° C. for a further 48 h with gentleinversion (9 rpm) of the sample tubes; digests are halted by heating(100° C., 5 min) and are then lyophilized; digests are resuspended in500 μL water and an aliquot (50 μL) is taken for analysis.

Capillary electrophoresis (CE) of disaccharides from digestion of HSpreparations may be conducted as follows: capillary electrophoresisoperating buffer is made by adding an aqueous solution of 20 mM H₃PO₄ toa solution of 20 mM Na₂HPO₄.12H₂O to give pH 3.5; column wash is 100 mMNaOH (diluted from 50% w/w NaOH); operating buffer and column wash areboth filtered using a filter unit fitted with 0.2 μm cellulose acetatemembrane filters; stock solutions of disaccharide Is (e.g. 12) areprepared by dissolving the disaccharides in water (1 mg/mL); calibrationcurves for the standards are determined by preparing a mix containingall standards containing 10 μg/100 μL of each disaccharide and adilution series containing 10, 5, 2.5, 1.25, 0.625, 0.3125 μg/100 μL isprepared; including 2.5 μg of internal standard (ΔUA,2S-GlcNCOEt,6S).The digests of HS are diluted (50 μL/mL) with water and the sameinternal standard is added (2.5 μg) to each sample. The solutions arefreeze-dried and re-suspended in water (1 mL). The samples are filteredusing PTFE hydrophilic disposable syringe filter units.

Analyses are performed using a capillary electrophoresis instrument onan uncoated fused silica capillary tube at 25° C. using 20 mM operatingbuffer with a capillary voltage of 30 kV. The samples are introduced tothe capillary tube using hydrodynamic injection at the cathodic (reversepolarity) end. Before each run, the capillary is flushed with 100 mMNaOH (2 min), with water (2 min) and pre-conditioned with operatingbuffer (5 min). A buffer replenishment system replaces the buffer in theinlet and outlet tubes to ensure consistent volumes, pH and ionicstrength are maintained. Water only blanks are run at both thebeginning, middle and end of the sample sequence. Absorbance ismonitored at 232 nm. All data is stored in a database and issubsequently retrieved and re-processed.

Duplicate or triplicate digests/analyses may be performed and thenormalized percentage of the disaccharides in the HS digest iscalculated as the mean average of the results for the analyses.

The HS/BMP2 also preferably exhibits high affinity binding to BMP2protein or the peptide of SEQ ID NO:1 or 6, as described in thisspecification.

The structural differences of HS/BMP2 compared with heparan sulphatesthat do not bind BMP2 protein are also illustrated by conducting surfaceplasmon resonance analysis. For example, the angle shift curveillustrated in FIG. 44 can be used to distinguish HS/BMP2 from otherheparan sulphates.

The structural differences of HS/BMP2 compared with heparan sulphatesthat do not bind BMP2 protein are further illustrated by conductingstrong anion exchange high pressure liquid chromatography (SAX-HPLC).FIG. 40 illustrates the SAX-HPLC spectrum of HS/BMP2, which may becompared with the SAX-HPLC spectra of heparan sulphates that do not bindBMP2 protein (FIGS. 41 and 42).

To identify HS/BMP2 the inventors devised a method that involvesenriching for glycosaminoglycan molecules that exhibit binding toparticular polypeptides having a heparin-binding domain. Isolated GAGmixtures and/or molecules can then be identified and tested for theirability to modulate the growth and differentiation of cells and tissueexpressing a protein containing the heparin-binding domain. This enablesthe controlled analysis of the effect of particular GAG saccharidesequences on the growth and differentiation of cells and tissue, both invitro and in vivo. The inventors applied this methodology to BoneMorphogenetic Protein 2 (BMP2) in order to isolate and characterise GAGshaving high binding to BMP2.

Accordingly, to identify HS/BMP2 the inventors provided a method ofisolating glycosaminoglycans capable of binding to proteins havingheparin/heparan-binding domains, the method comprising:

-   -   (i) providing a solid support having polypeptide molecules        adhered to the support, wherein the polypeptide comprises a        heparin-binding domain;    -   (ii) contacting the polypeptide molecules with a mixture        comprising glycosaminoglycans such that        polypeptide-glycosaminoglycan complexes are allowed to form;    -   (iii) partitioning polypeptide-glycosaminoglycan complexes from        the remainder of the mixture;    -   (iv) dissociating glycosaminoglycans from the        polypeptide-glycosaminoglycan complexes;    -   (v) collecting the dissociated glycosaminoglycans.

The inventors also provided isolated glycosaminoglycans identified bytheir ability to modulate the growth or differentiation of cells ortissues. To do this, they provided a method of identifyingglycosaminoglycans capable of stimulating or inhibiting the growthand/or differentiation of cells and/or tissues, the method comprising:

-   -   (i) providing a solid support having polypeptide molecules        adhered to the support, wherein the polypeptide comprises a        heparin-binding domain;    -   (ii) contacting the polypeptide molecules with a mixture        comprising glycosaminoglycans such that        polypeptide-glycosaminoglycan complexes are allowed to form;    -   (iii) partitioning polypeptide-glycosaminoglycan complexes from        the remainder of the mixture;    -   (iv) dissociating glycosaminoglycans from the        polypeptide-glycosaminoglycan complexes;    -   (v) collecting the dissociated glycosaminoglycans;    -   (vi) adding the collected glycosaminoglycans to cells or tissues        in which a protein containing the amino acid sequence of the        heparin-binding domain is present;    -   (vii) measuring one or more of: proliferation of the cells,        differentiation of the cells, expression of one or more protein        markers.

The inventors used these methods to identify a GAG capable of binding toBMP2 (which they called HS/BMP2 or HS3 or HS-3), wherein the polypeptideused in the inventors' methodology comprised the heparin-binding domainof SEQ ID NO:1 or 6, derived from the amino acid sequence of BMP2.

In the inventors' methodology, the mixture comprising GAGs may containsynthetic glycosaminoglycans. However, GAGs obtained from cells ortissues are preferred. For example, the mixture may containextracellular matrix wherein the extracellular matrix material isobtained by scraping live tissue in situ (i.e. directly from the tissuein the body of the human or animal from which it is obtained) or byscraping tissue (live or dead) that has been extracted from the body ofthe human or animal. Alternatively, the extracellular matrix materialmay be obtained from cells grown in culture. The extracellular matrixmaterial may be obtained from connective tissue or connective tissuecells, e.g. bone, cartilage, muscle, fat, ligament or tendon. Forisolation of HS/BMP2 one suitable source of extracellular matrixmaterial are osteoblasts, such as mouse MC3T3 cells.

The GAG component may be extracted from a tissue or cell sample orextract by a series of routine separation steps (e.g. anion exchangechromatography), well known to those of skill in the art.

GAG mixtures may contain a mixture of different types ofglycosaminoglycan, which may include dextran sulphates, chondroitinsulphates and heparan sulphates. Preferably, the GAG mixture contactedwith the solid support is enriched for heparan sulphate. A heparansulphate-enriched GAG fraction may be obtained by performing columnchromatography on the GAG mixture, e.g. weak, medium or strong anionexchange chromatography, as well as strong high pressure liquidchromatography (SAX-HPLC), with selection of the appropriate fraction.

The collected GAGs may be subjected to further analysis in order toidentify the GAG, e.g. determine GAG composition or sequence, ordetermine structural characteristics of the GAG. GAG structure istypically highly complex, and, taking account of currently availableanalytical techniques, exact determinations of GAG sequence structureare not possible in most cases.

However, the collected GAG molecules may be subjected to partial orcomplete saccharide digestion (e.g. chemically by nitrous acid orenzymatically with lyases such as heparinase III) to yield saccharidefragments that are both characteristic and diagnostic of the GAG. Inparticular, digestion to yield disaccharides (or tetrasaccharides) maybe used to measure the percentage of each disaccharide obtained whichwill provide a characteristic disaccharide “fingerprint” of the GAG.

The pattern of sulphation of the GAG can also be determined and used todetermine GAG structure. For example, for heparan sulphate the patternof sulphation at amino sugars and at the C2, C3 and C6 positions may beused to characterise the heparan sulphate.

Disaccharide analysis, tetrasaccharide analysis and analysis ofsulphation can be used in conjunction with other analytical techniquessuch as HPLC, mass spectrometry and NMR which can each provide uniquespectra for the GAG. In combination, these techniques may provide adefinitive structural characterisation of the GAG.

A high affinity binding interaction between the GAG and heparin-bindingdomain indicates that the GAG will contain a specific saccharidesequence that contributes to the high affinity binding interaction. Afurther step may comprise determination of the complete or partialsaccharide sequence of the GAG, or the key portion of the GAG, involvedin the binding interaction.

GAG-polypeptide complexes may be subjected to treatment with an agentthat lyses glycosaminoglycan chains, e.g. a lyase. Lyase treatment maycleave portions of the bound GAG that are not taking part in the bindinginteraction with the polypeptide. Portions of the GAG that are takingpart in the binding interaction with the polypeptide may be protectedfrom lyase action. After removal of the lyase, e.g. following a washingstep, the GAG molecule that remains bound to the polypeptide representsthe specific binding partner (“GAG ligand”) of the polypeptide. Owing tothe lower complexity of shorter GAG molecules, following dissociationand collection of the GAG ligand, a higher degree of structuralcharacterisation of the GAG ligand can be expected. For example, thecombination of any of the saccharide sequence (i.e. the primary (linear)sequence of monosaccharides contained in the GAG ligand), sulphationpattern, disaccharide and/or tetrasaccharide digestion analysis, NMRspectra, mass spectrometry spectra and HPLC spectra may provide a highlevel of structural characterisation of the GAG ligand.

As used herein, the terms ‘enriching’, ‘enrichment’, ‘enriched’, etc.describes a process (or state) whereby the relative composition of amixture is (or has been) altered in such a way that the fraction of thatmixture given by one or more of those entities is increased, while thefraction of that mixture given by one or more different entities isdecreased.

GAGs isolated by enrichment may be pure, i.e. contain substantially onlyone type of GAG, or may continue to be a mixture of different types ofGAG, the mixture having a higher proportion of particular GAGs that bindto the heparin-binding domain relative to the starting mixture.

The GAGs identified preferably exhibit a functional effect whencontacted with cells or tissue in which a protein containing theheparin-binding domain is expressed or contained. The functional effectmay be a modulating or potentiating effect.

The functional effect may be to promote (stimulate) or inhibit theproliferation of the cells of a certain type or the differentiation ofone cell type into another, or the expression of one or more proteinmarkers. For example, the GAGs may promote cell proliferation, i.e. anincrease in cell number, or promote differentiation of stem cells intospecialised cell types (e.g. mesenchymal stem cells in connectivetissue), promote or inhibit the expression of protein markers indicativeof the multipotency or differentiation state of the cells (e.g. markerssuch as alkaline phosphatase activity, detection of RUNX2, osterix,collagen I, II, IV, VII, X, osteopontin, Osteocalcin, BSPII, SOX9,Aggrecan, ALBP, CCAAT/enhancer binding protein-α (C/EBPα), adipocytelipid binding protein (ALBP), alkaline phosphatase (ALP), bonesialoprotein 2, (BSPII), Collagen2a1 (Coll2a) and SOX9).

As used herein, the term ‘modulating effect’ is understood to mean theeffect that a first entity has on a second entity wherein the secondentity's normal function in another process or processes is modified bythe presence of the first entity. The modulating effect may be eitheragonistic or antagonistic.

The modulating effect may be a potentiating effect. The term‘potentiating effect’ is understood to mean the effect of increasingpotency. In a preferred embodiment of the present invention, the term‘potentiating effect’ refers to the effect that a first entity has on asecond entity, which effect increases the potency of that second entityin another process or processes. In a further preferred embodiment ofthe present invention, the potentiating effect is understood to mean theeffect of isolated GAGs on a heparin-binding factor, wherein the saideffect increases the potency of said heparin-binding factor.

The potentiating effect may be an increase in bioavailability of theheparin-binding factor. In a preferred embodiment of the presentinvention, the potentiating effect is an increase in bioavailability ofBMP2. One method of measuring an increase in bioavailability of theheparin-binding factor is through determining an increase in localconcentration of the heparin-binding factor.

The potentiating effect may be to protect the heparin-binding factorfrom degradation. In an especially preferred embodiment of the presentinvention, the potentiating effect is to protect BMP-2 from degradation.One method of determining a decrease in the degradation of theheparin-binding factor is through measuring an increase in the half-lifeof the heparin-binding factor.

The potentiating effect may be to sequester heparin-binding factors awayfrom cellular receptors or may be to stabilise the ligand-receptorinteraction.

The potentiating effect (e.g. modulation of growth or differentiation)may be determined by use of appropriate assays. For example, the effectthat an HS has on the stability of BMP-2 may be determined by ELISA. Theeffect that an HS has on the activity of BMP-2 may be determined bymeasuring the activation/expression of one or more of SMAD 1, 5 or 8, ormeasuring the expression of one or more osteogenic marker genes such asRunx2, alkaline phosphatase, Osterix, Osteocalcin and BSP1, or measuringthe levels of mineralization using staining such as Alizarin Red and vonKossa.

As used herein, the process of ‘contacting’ involves the bringing intoclose physical proximity of two or more discrete entities. The processof ‘contacting’ involves the bringing into close proximity of two ormore discrete entities for a time, and under conditions, sufficient toallow a portion of those two or more discrete entities to interact on amolecular level. Preferably, as used herein, the process of ‘contacting’involves the bringing into close proximity of the mixture of compoundspossessing one or more GAGs and the polypeptide corresponding to theheparin-binding domain of a heparin-binding factor. Examples of‘contacting’ processes include mixing, dissolving, swelling, washing. Inpreferred embodiments ‘contact’ of the GAG mixture and polypeptide issufficient for complexes, which may be covalent but are preferablynon-covalent, to form between GAGs and polypeptides that exhibit highaffinity for each other.

The polypeptide may comprise the full length or near full length primaryamino acid sequence of a selected protein having a heparin-bindingdomain. Due to folding that may occur in longer polypeptides leading topossible masking of the heparin-binding domain from the GAG mixture, itis preferred for the polypeptide to be short. Preferably, thepolypeptide will have an amino acid sequence that includes theheparin-binding domain and optionally including one or more amino acidsat one or each of the N- and C-terminals of the peptides. Theseadditional amino acids may enable the addition of linker or attachmentmolecules to the polypeptide that are required to attach the polypeptideto the solid support.

In preferred embodiments of the inventors' methodology, in addition tothe number of amino acids in the heparin-binding domain the polypeptidecontains 1-20, more preferably 1-10, still more preferably 1-5additional amino acids. In some embodiments the amino acid sequence ofthe heparin-binding domain accounts for at least 80% of the amino acidsof the polypeptide, more preferably at least 90%, still more preferablyat least 95%.

In order to adhere polypeptides to the surface of a solid support thepolypeptides are preferably modified to include a molecular tag, and thesurface of the solid support is modified to incorporate a correspondingmolecular probe having high affinity for the molecular tag, i.e. themolecular tag and probe form a binding pair. The tag and/or probe may bechosen from any one of: an antibody, a cell receptor, a ligand, biotin,any fragment or derivative of these structures, any combination of theforegoing, or any other structure with which a probe can be designed orconfigured to bind or otherwise associate with specificity. A preferredbinding pair suitable for use as tag and probe is biotin and avidin.

The polypeptide is derived from the protein of interest, which in thepresent case is BMP2. By “derived from” is meant that the polypeptide ischosen, selected or prepared because it contains the amino acid sequenceof a heparin-binding domain that is present in the protein of interest.The amino acid sequence of the heparin-binding domain may be modifiedfrom that appearing in the protein of interest, e.g. to investigate theeffect of changes in the heparin-binding domain sequence on GAG binding.

In this specification the protein is BMP2. The amino acid sequence ofthe preferred heparin-binding domain from BMP2 is QAKHKQRKRLKSSCKRHP(SEQ ID NO:1), which is found at amino acids 283-300 of SEQ ID NO:2(FIG. 35) or QAKHKQRKRLKSSCKRH (SEQ ID NO:6) which is found at aminoacids 283-299 of SEQ ID NO:2.

It is understood by those skilled in the art that small variations inthe amino acid sequence of a particular polypeptide may allow theinherent functionality of that portion to be maintained. It is alsounderstood that the substitution of certain amino acid residues within apeptide with other amino acid residues that are isosteric and/orisoelectronic may either maintain or improve certain properties of theunsubstituted peptide. These variations are also encompassed within thescope of the present invention. For example, the amino acid alanine maysometimes be substituted for the amino acid glycine (and vice versa)whilst maintaining one or more of the properties of the peptide. Theterm ‘isosteric’ refers to a spatial similarity between two entities.Two examples of moieties that are isosteric at moderately elevatedtemperatures are the iso-propyl and tert-butyl groups. The term‘isoelectronic’ refers to an electronic similarity between two entities,an example being the case where two entities possess a functionality ofthe same, or similar, pKa.

The polypeptide corresponding to the heparin-binding domain may besynthetic or recombinant.

The solid support may be any substrate having a surface to whichmolecules may be attached, directly or indirectly, through eithercovalent or non-covalent bonds. The solid support may include anysubstrate material that is capable of providing physical support for theprobes that are attached to the surface. It may be a matrix support. Thematerial is generally capable of enduring conditions related to theattachment of the probes to the surface and any subsequent treatment,handling, or processing encountered during the performance of an assay.The materials may be naturally occurring, synthetic, or a modificationof a naturally occurring material. The solid support may be a plasticsmaterial (including polymers such as, e.g., poly(vinyl chloride),cyclo-olefin copolymers, polyacrylamide, polyacrylate, polyethylene,polypropylene, poly(4-methylbutene), polystyrene, polymethacrylate,poly(ethylene terephthalate), polytetrafluoroethylene (PTFE or Teflon®),nylon, poly(vinyl butyrate)), etc., either used by themselves or inconjunction with other materials. Additional rigid materials may beconsidered, such as glass, which includes silica and further includes,for example, glass that is available as Bioglass. Other materials thatmay be employed include porous materials, such as, for example,controlled pore glass beads. Any other materials known in the art thatare capable of having one or more functional groups, such as any of anamino, carboxyl, thiol, or hydroxyl functional group, for example,incorporated on its surface, are also contemplated.

Preferred solid supports include columns having a polypeptideimmobilized on a surface of the column. The surface may be a wall of thecolumn, and/or may be provided by beads packed into the central space ofthe column.

The polypeptide may be immobilised on the solid support. Examples ofmethods of immobilisation include: adsorption, covalent binding,entrapment and membrane confinement. In a preferred embodiment of thepresent invention the interaction between the polypeptide and the matrixis substantially permanent. In a further preferred embodiment of thepresent invention, the interaction between the peptide and the matrix issuitably inert to ion-exchange chromatography. In a preferredarrangement, the polypeptide is attached to the surface of the solidsupport. It is understood that a person skilled in the art would have alarge array of options to choose from to chemically and/or physicallyattach two entities to each other. These options are all encompassedwithin the scope of the present invention. In a preferred arrangement,the polypeptide is adsorbed to a solid support through the interactionof biotin with streptavidin. In a representative example of thisarrangement, a molecule of biotin is bonded covalently to thepolypeptide, whereupon the biotin-polypeptide conjugate binds tostreptavidin, which in turn has been covalently bonded to a solidsupport. In another arrangement, a spacer or linker moiety may be usedto connect the molecule of biotin with the polypeptide, and/or thestreptavidin with the matrix.

By contacting the GAG mixture with the solid support GAG-polypeptidecomplexes are allowed to form. These are partitioned from the remainderof the mixture by removing the remainder of the mixture from the solidsupport, e.g. by washing the solid support to elute non-bound materials.Where a column is used as the solid support non-binding components ofthe GAG mixture can be eluted from the column leaving theGAG-polypeptide complexes bound to the column.

It is understood that certain oligosaccharides may interact in anon-specific manner with the polypeptide. In certain embodiments,oligosaccharide which interacts with the polypeptide in a non-specificmanner may be included in, or excluded from the mixture of compoundsenriched with one or more GAGs that modulate the effect of aheparin-binding factor. An example of a non-specific interaction is thetemporary confinement within a pocket of a suitably sized and/or shapedmolecule. Further it is understood that these oligosaccharides may elutemore slowly than those oligosaccharides that display no interaction withthe peptide at all. Furthermore it is understood that the compounds thatbind non-specifically may not require the input of the same externalstimulus to make them elute as for those compounds that bind in aspecific manner (for example through an ionic interaction). Theinventors' methodology is capable of separating a mixture ofoligosaccharides into those components of that mixture that: bind in aspecific manner to the polypeptide; those that bind in a non-specificmanner to the polypeptide; and those that do not bind to thepolypeptide. These designations are defined operationally for eachGAG-peptide pair.

By varying the conditions (e.g. salt concentration) present at thesurface of the solid support where binding of the GAG and polypeptideoccurs those GAGs having the highest affinity and/or specificity for theheparin-binding domain can be selected.

GAGs may accordingly be obtained that have a high binding affinity for aprotein of interest and/or the heparin-binding domain of the protein ofinterest. The binding affinity (K_(d)) may be chosen from one of: lessthan 10 μM, less than 1 μM, less than 100 nM, less than 10 nM, less than1 nM, less than 100 μM.

GAGs obtained by the methods described may be useful in a range ofapplications, in vitro and/or in vivo. The GAGs may be provided for usein stimulation or inhibition of cell or tissue growth and/orproliferation and/or differentiation either in cell or tissue culture invitro, or in cells or tissue in vivo.

The GAGs may be provided as a formulation for such purposes. Forexample, culture media may be provided comprising a GAG obtained by themethod described, i.e. comprising HS/BMP2.

Cells or tissues obtained from in vitro cell or tissue culture in thepresence of HS/BMP2 may be collected and implanted into a human oranimal patient in need of treatment. A method of implantation of cellsand/or tissues may therefore be provided, the method comprising thesteps of:

-   -   (a) culturing cells and/or tissues in vitro in contact with        HS/BMP2;    -   (b) collecting the cells and/or tissues;    -   (c) implanting the cells and/or tissues into a human or animal        subject in need of treatment.

The cells may be cultured in part (a) in contact with HS/BMP2 for aperiod of time sufficient to allow growth, proliferation ordifferentiation of the cells or tissues. For example, the period of timemay be chosen from: at least 5 days, at least 10 days, at least 20 days,at least 30 days or at least 40 days.

In another embodiment the HS/BMP2 may be formulated for use in a methodof medical treatment, including the prevention or treatment of injury ordisease. A pharmaceutical composition or medicament may be providedcomprising HS/BMP2 and a pharmaceutically acceptable diluent, carrier oradjuvant. Such pharmaceutical compositions or medicaments may beprovided for the prevention or treatment of injury or disease. The useof HS/BMP2 in the manufacture of a medicament for the prevention ortreatment of injury or disease is also provided. Optionally,pharmaceutical compositions and medicaments according to the presentinvention may also contain the protein of interest (i.e. BMP2) havingthe heparin-binding domain to which the GAG binds. In furtherembodiments the pharmaceutical compositions and medicaments may furthercomprise stem cells, e.g. mesenchymal stem cells.

Treatment of injury or disease may comprise the repair, regeneration orreplacement of cells or tissue, such as connective tissue (e.g. bone,cartilage, muscle, fat, tendon or ligament). For the repair of tissue,the pharmaceutical composition or medicament comprising HS/BMP2 may beadministered directly to the site of injury or disease in order tostimulate the growth, proliferation and/or differentiation of new tissueto effect a repair of the injury or to cure or alleviate (e.g. providerelief to the symptoms of) the disease condition. The repair orregeneration of the tissue may be improved by combining stem cells inthe pharmaceutical composition or medicament.

For the replacement of tissue, HS/BMP2 may be contacted with cellsand/or tissue during in vitro culture of the cells and/or tissue inorder to generate cells and/or tissue for implantation at the site ofinjury or disease in the patient. Implantation of cells or tissue can beused to effect a repair of the injured or diseased tissue in the patientby replacement of the injured or diseased tissue. This may involveexcision of injured/diseased tissue and implantation of new tissueprepared by culture of cells and/or tissue in contact with HS/BMP2.

Pharmaceutical compositions and medicaments according to the presentinvention may therefore comprise one of:

-   -   (a) HS/BMP2;    -   (b) HS/BMP2 in combination with stem cells;    -   (c) HS/BMP2 in combination with a protein containing the        heparin-binding domain bound by HS/BMP2 (i.e. SEQ ID NO:1 or 6);    -   (d) HS/BMP2 in combination with stem cells and a protein        containing the heparin-binding domain bound by HS/BMP2 (i.e. SEQ        ID NO:1 or 6);    -   (e) Tissues or cells obtained from culture of cells or tissues        in contact with HS/BMP2.

HS/BMP2 may be used in the repair or regeneration of bodily tissue,especially bone regeneration, neural regeneration, skeletal tissueconstruction, the repair of cardio-vascular injuries and the expansionand self-renewal of embryonic and adult stem cells. Accordingly, HS/BMP2may be used to prevent or treat a wide range of diseases and injuries,including osteoarthritis, cartilage replacement, broken bones of anykind (e.g. spinal disc fusion treatments, long bone breaks, cranialdefects), critical or non-union bone defect regeneration.

The use of HS/BMP2 in the repair, regeneration or replacement of tissuemay involve use in wound healing, e.g. acceleration of wound healing,healing of scar or bone tissue and tissue grafting.

In another aspect, the present invention provides a biological scaffoldcomprising HS/BMP2. In some embodiments, the biological scaffolds of thepresent invention may be used in orthopaedic, vascular, prosthetic, skinand corneal applications. The biological scaffolds provided by thepresent invention include extended-release drug delivery devices, tissuevalves, tissue valve leaflets, drug-eluting stents, vascular grafts,wound healing or skin grafts and orthopaedic prostheses such as bone,ligament, tendon, cartilage and muscle. In a preferred embodiment of thepresent invention, the biological scaffold is a catheter wherein theinner (and/or outer) surface comprises one or more GAG compounds(including HS/BMP2) attached to the catheter.

In another aspect, the present invention provides one or more GAGs(including HS/BMP2) isolated by the method described for use as anadjuvant. The adjuvant may be an immune adjuvant.

In another aspect, the present invention provides pharmaceuticallyacceptable formulations comprising a mixture of compounds comprising oneor more GAGs, said mixture being enriched with respect to HS/BMP2. Inanother aspect, the invention provides pharmaceutically acceptableformulations comprising:

-   -   (i) a mixture of compounds comprising one or more GAGs, said        mixture being enriched with respect to HS/BMP2; and    -   (ii) BMP-2,        for separate, simultaneous or sequential administration. In a        preferred embodiment the formulation comprises the mixture of        compounds comprising one or more GAGs, said mixture being        enriched with respect to HS/BMP2 and BMP-2 in intimate        admixture, and is administered simultaneously to a patient in        need of treatment.

In another aspect of the present invention a kit is provided for use inthe repair, or regeneration of tissue, said kit comprising (i) apredetermined amount of HS/BMP2, and (ii) a predetermined amount ofBMP2.

The compounds of the enriched mixtures of the present invention can beadministered to a subject as a pharmaceutically acceptable salt thereof.For example, base salts of the compounds of the enriched mixtures of thepresent invention include, but are not limited to, those formed withpharmaceutically acceptable cations, such as sodium, potassium, lithium,calcium, magnesium, ammonium and alkylammonium. The present inventionincludes within its scope cationic salts, for example the sodium orpotassium salts.

It will be appreciated that the compounds of the enriched mixtures ofthe present invention which bear a carboxylic acid group may bedelivered in the form of an administrable prodrug, wherein the acidmoiety is esterified (to have the form —CO2R′). The term “pro-drug”specifically relates to the conversion of the —OR′ group to a —OH group,or carboxylate anion therefrom, in vivo. Accordingly, the prodrugs ofthe present invention may act to enhance drug adsorption and/or drugdelivery into cells. The in vivo conversion of the prodrug may befacilitated either by cellular enzymes such as lipases and esterases orby chemical cleavage such as in vivo ester hydrolysis.

Medicaments and pharmaceutical compositions according to aspects of thepresent invention may be formulated for administration by a number ofroutes, including but not limited to, injection at the site of diseaseor injury. The medicaments and compositions may be formulated in fluidor solid form. Fluid formulations may be formulated for administrationby injection to a selected region of the human or animal body.

Administration is preferably in a “therapeutically effective amount”,this being sufficient to show benefit to the individual. The actualamount administered, and rate and time-course of administration, willdepend on the nature and severity of the injury or disease beingtreated. Prescription of treatment, e.g. decisions on dosage etc, iswithin the responsibility of general practitioners and other medicaldoctors, and typically takes account of the disorder to be treated, thecondition of the individual patient, the site of delivery, the method ofadministration and other factors known to practitioners. Examples of thetechniques and protocols mentioned above can be found in Remington'sPharmaceutical Sciences, 20th Edition, 2000, pub. Lippincott, Williams &Wilkins.

In this specification a patient to be treated may be any animal orhuman. The patient may be a non-human mammal, but is more preferably ahuman patient. The patient may be male or female.

Methods according to the present invention may be performed in vitro orin vivo, as indicated. The term “in vitro” is intended to encompassprocedures with cells in culture whereas the term “in vivo” is intendedto encompass procedures with intact multi-cellular organisms.

Stem Cells

Cells contacted with HS/BMP2 include stem cells.

The stem cells cultured and described herein may be stem cells of anykind. They may be totipotent or multipotent (pluripotent). They may beembryonic or adult stem cells from any tissue and may be hematopoieticstem cells, neural stem cells or mesenchymal stem cells. Preferably theyare adult stem cells. More preferably they are adult mesenchymal stemcells, e.g. capable of differentiation into connective tissue and/orbone cells such as chondrocytes, osteoblasts, myocytes and adipocytes.The stem cells may be obtained from any animal or human, e.g. non-humananimals, e.g. rabbit, guinea pig, rat, mouse or other rodent (includingcells from any animal in the order Rodentia), cat, dog, pig, sheep,goat, cattle, horse, non-human primate or other non-human vertebrateorganism; and/or non-human mammalian animals; and/or human. Optionallythey are non-human.

In this specification, by stem cell is meant any cell type that has theability to divide (i.e. self-renew) and remain totipotent or multipotent(pluripotent) and give rise to specialized cells if so desired.

Stem cells cultured in the present invention may be obtained or derivedfrom existing cultures or directly from any adult, embryonic or fetaltissue, including blood, bone marrow, skin, epithelia or umbilical cord(a tissue that is normally discarded).

The multipotency of stem cells may be determined by use of suitableassays. Such assays may comprise detecting one or more markers ofpluripotency, e.g. alkaline phosphatase activity, detection of RUNX2,osterix, collagen I, II, IV, VII, X, osteopontin, Osteocalcin, BSPII,SOX9, Aggrecan, ALBP, CCAAT/enhancer binding protein-α (C/EBPα),adipocyte lipid binding protein (ALBP), alkaline phosphatase (ALP), bonesialoprotein 2, (BSPII), Collagen2a1 (Coll2a) and SOX9.

Mesenchymal stem cells or human bone marrow stromal stem cells aredefined as pluripotent (multipotent) progenitor cells with the abilityto generate cartilage, bone, muscle, tendon, ligament and fat. Theseprimitive progenitors exist postnatally and exhibit stem cellcharacteristics, namely low incidence and extensive renewal potential.These properties in combination with their developmental plasticity havegenerated tremendous interest in the potential use of mesenchymal stemcells to replace damaged tissues. In essence mesenchymal stem cellscould be cultured to expand their numbers then transplanted to theinjured site or after seeding in/on scaffolds to generate appropriatetissue constructs.

Thus, an alternative approach for skeletal, muscular, tendon andligament repair is the selection, expansion and modulation of theappropriate progenitor cells such as osteoprogenitor cells in the caseof bone in combination with a conductive or inductive scaffolds tosupport and guide regeneration together with judicious selection ofspecific tissue growth factors.

Human bone marrow mesenchymal stem cells can be isolated and detectedusing selective markers, such as STRO-I, from a CD34+ fractionindicating their potential for marrow repopulation. These cell surfacemarkers are only found on the cell surface of mesenchymal stem cells andare an indication of the cells pluripotency.

Mesenchymal cells are easily obtainable from bone marrow by minimallyinvasive techniques and can be expanded in culture and permitted todifferentiate into the desired lineage. Differentiation can be inducedby the application of specific growth factors. The transforming growthfactor beta (TGF-beta) superfamily member proteins such as the bonemorphogenetic proteins (BMPs) are important factors of chondrogenic andosteogenic differentiation of mesenchymal stem cells.

Suitable MSCs may be obtained or derived from bone marrow mononuclearcells (BMMNCs) collected from aspirates of bone marrow (e.g. Wexler etal. Adult bone marrow is a rich source of human mesenchymal ‘stem’ cellsbut umbilical cord and mobilized adult blood are not. HAEMOPOIESIS ANDLEUCOCYTES British Journal of Haematology 121(2):368-374, April 2003.)or Wharton's Jelly of the umbilical cord (e.g. Ta et al. Long-termExpansion and Pluripotent Marker Array Analysis of Wharton'sJelly-Derived Mesenchymal Stem Cells. Stem Cells Dev. 2009 Jul. 20(Epub)).

Mesenchymal stem cells may be obtained by differentiation of pluripotentstem cells, such as human embryonic stem cells or induced pluripotentstem cells, by application of suitable differentiating factors, as iswell known in the art.

In yet a further aspect of the present invention, a pharmaceuticalcomposition comprising stem cells generated by any of the methods of thepresent invention, or fragments or products thereof, is provided. Thepharmaceutical composition useful in a method of medical treatment.Suitable pharmaceutical compositions may further comprise apharmaceutically acceptable carrier, adjuvant or diluent.

In another aspect of the present invention, stem cells generated by anyof the methods of the present invention may be used in a method ofmedical treatment, preferably, a method of medical treatment is providedcomprising administering to an individual in need of treatment atherapeutically effective amount of said medicament or pharmaceuticalcomposition.

Stem cells obtained through culture methods and techniques according tothis invention may be used to differentiate into another cell type foruse in a method of medical treatment. Thus, the differentiated cell typemay be derived from, and may be considered as a product of, a stem cellobtained by the culture methods and techniques described which hassubsequently been permitted to differentiate. Pharmaceuticalcompositions may be provided comprising such differentiated cells,optionally together with a pharmaceutically acceptable carrier, adjuvantor diluent. Such pharmaceutical composition may be useful in a method ofmedical treatment.

Glycosaminglycans

As used herein, the terms ‘glycosaminoglycan’ and ‘GAG’ are usedinterchangeably and are understood to refer to the large collection ofmolecules comprising an oligosaccharide, wherein one or more of thoseconjoined saccharides possess an amino substituent, or a derivativethereof. Examples of GAGs are chondroitin sulfate, keratan sulfate,heparin, dermatan sulfate, hyaluronate and heparan sulfate. Heparansulfates are preferred embodiments of the present invention.

As used herein, the term ‘GAG’ also extends to encompass those moleculesthat are GAG conjugates. An example of a GAG conjugate is aproteoglycosaminoglycan (PGAG, proteoglycan) wherein a peptidiccomponent is covalently bound to an oligosaccharide component.

In the present invention, it is understood that there are a large numberof sources of GAG compounds including natural, synthetic orsemi-synthetic. A preferred source of GAGs is biological tissue. Apreferred source of GAGs is a stem cell. An especially preferred sourceof GAGs is a stem cell capable of differentiating into a cell thatcorresponds to a tissue that will be the subject of treatment. Forexample, GAGs can be sourced from preosteoblasts for use in boneregeneration or skeletal tissue construction. In an especially preferredembodiment of the present invention, GAGs may be sourced from animmortalised cell line. In a further preferred embodiment of the presentinvention, GAGs may be sourced from an immortalised cell line which isgrown in a bioreactor. Another preferred source of GAGs is a syntheticsource. In this respect, GAGs may be obtained from the syntheticelaboration of commercially available starting materials into morecomplicated chemical form through techniques known, or conceivable, toone skilled in the art. An example of such a commercially availablestarting material is glucosamine. Another preferred source of GAGs is asemi-synthetic source. In this respect, synthetic elaboration of anatural starting material, which possesses much of the complexity of thedesired material, is elaborated synthetically using techniques known, orconceivable, to one skilled in the art. Examples of such a naturalstarting material are chitin and dextran, and examples of the types ofsynthetic steps that may elaborate that starting material, into a GAGmixture suitable for use in the present invention, are amide bondhydrolysis, oxidation and sulfation. Another example of a semi-syntheticroute to GAGs of the desired structure comprises the syntheticinterconversion of related GAGs to obtain GAGs suitable for use in thepresent invention.

Heparan Sulphate (HS)

In preferred aspects of the invention the glycosaminoglycan orproteoglycan is preferably a heparan sulfate.

Heparan sulfate proteoglycans (HSPGs) represent a highly diversesubgroup of proteoglycans and are composed of heparan sulfateglycosaminoglycan side chains covalently attached to a protein backbone.The core protein exists in three major forms: a secreted form known asperlecan, a form anchored in the plasma membrane known as glypican, anda transmembrane form known as syndecan. They are ubiquitous constituentsof mammalian cell surfaces and most extracellular matrices. There areother proteins such as agrin, or the amyloid precursor protein, in whichan HS chain may be attached to less commonly found cores.

“Heparan Sulphate” (“Heparan sulfate” or “HS”) is initially synthesisedin the Golgi apparatus as polysaccharides consisting of tandem repeatsof D-glucuronic acid (GlcA) and N-acetyl-D-glucosamine (GlcNAc). Thenascent polysaccharides may be subsequently modified in a series ofsteps: N-deacetylation/N-sulfation of GlcNAc, C5 epimerisation of GlcAto iduronic acid (IdoA), O-sulphation at C2 of IdoA and GlcA, O—sulphation at C6 of N-sulphoglucosamine (GlcNS) and occasionalO-sulphation at C3 of GlcNS. N-deacetylation/N-sulphation, 2-O—, 6-O—and 3-O-sulphation of HS are mediated by the specific action of HSN-deacetylase/N-sulfotransferase (HSNDST), HS 2-O— sulfotransferase(HS2ST), HS 6-O-sulfotransferase (HS6ST) and HS 3-O-sulfotransferase,respectively. At each of the modification steps, only a fraction of thepotential substrates are modified, resulting in considerable sequencediversity. This structural complexity of HS has made it difficult todetermine its sequence and to understand the relationship between HSstructure and function.

Heparan sulfate side chains consist of alternately arranged D-glucuronicacid or L-iduronic acid and D-glucosamine, linked via (1->4) glycosidicbonds. The glucosamine is often N-acetylated or N-sulfated and both theuronic acid and the glucosamine may be additionally O-sulfated. Thespecificity of a particular HSPG for a particular binding partner iscreated by the specific pattern of carboxyl, acetyl and sulfate groupsattached to the glucosamine and the uronic acid. In contrast to heparin,heparan sulfate contains less N- and O-sulfate groups and more N-acetylgroups. The heparan sulfate side chains are linked to a serine residueof the core protein through a tetrasaccharide linkage(-glucuronosyl-β-(1→3)-galactosyl-β-(1→3)-galactosyl-β-(1→4)-xylosyl-→-1-O-(Serine))region.

Both heparan sulfate chains and core protein may undergo a series ofmodifications that may ultimately influence their biological activity.Complexity of HS has been considered to surpass that of nucleic acids(Lindahl et al, 1998, J. Biol. Chem. 273, 24979; Sugahara and Kitagawa,2000, Curr. Opin. Struct. Biol. 10, 518). Variation in HS species arisesfrom the synthesis of non-random, highly sulfated sequences of sugarresidues which are separated by unsulfated regions of disaccharidescontaining N— acetylated glucosamine. The initial conversion ofN-acetylglucosamine to N— sulfoglucosamine creates a focus for othermodifications, including epimerization of glucuronic acid to iduronicacid and a complex pattern of O-sulfations on glucosamine or iduronicacids. In addition, within the non-modified, low sulfated, N-acetylatedsequences, the hexuronate residues remain as glucuronate, whereas in thehighly sulfated N-sulfated regions, the C-5 epimer iduronatepredominates. This limits the number of potential disaccharide variantspossible in any given chain but not the abundance of each. Mostmodifications occur in the N-sulfated domains, or directly adjacent tothem, so that in the mature chain there are regions of high sulfationseparated by domains of low sulfation (Brickman et al. (1998), J. Biol.Chem. 273(8), 4350-4359, which is herein incorporated by reference inits entirety).

It is hypothesized that the highly variable heparan sulfate chains playkey roles in the modulation of the action of a large number ofextracellular ligands, including regulation and presentation of growthand adhesion factors to the cell, via a complicated combination ofautocrine, juxtacrine and paracrine feedback loops, so controllingintracellular signaling and thereby the differentiation of stem cells.For example, even though heparan sulfate glycosaminoglycans may begenetically described (Alberts et al. (1989) Garland Publishing, Inc,New York & London, pp. 804 and 805), heparan sulfate glycosaminoglycanspecies isolated from a single source may differ in biological activity.As shown in Brickman et al, 1998, Glycobiology 8, 463, two separatepools of heparan sulfate glycosaminoglycans obtained fromneuroepithelial cells could specifically activate either FGF-1 or FGF-2,depending on mitogenic status. Similarly, the capability of a heparansulfate (HS) to interact with either FGF-1 or FGF-2 is described in WO96/23003. According to this patent application, a respective HS capableof interacting with FGF-1 is obtainable from murine cells at embryonicday from about 11 to about 13, whereas a HS capable of interacting withFGF-2 is obtainable at embryonic day from about 8 to about 10.

As stated above HS structure is highly complex and variable between HS.Indeed, the variation in HS structure is considered to play an importantpart in contributing toward the different activity of each HS inpromoting cell growth and directing cell differentiation. The structuralcomplexity is considered to surpass that of nucleic acids and althoughHS structure may be characterised as a sequence of repeatingdisaccharide units having specific and unique sulfation patterns at thepresent time no standard sequencing technique equivalent to thoseavailable for nucleic acid sequencing is available for determining HSsequence structure. In the absence of simple methods for determining adefinitive HS sequence structure HS molecules are positively identifiedand structurally characterised by skilled workers in the field by anumber of analytical techniques. These include one or a combination ofdisaccharide analysis, tetrasaccharide analysis, HPLC and molecularweight determination. These analytical techniques are well known to andused by those of skill in the art.

Two techniques for production of di- and tetra-saccharides from HSinclude nitrous acid digestion and lyase digestion. A description of oneway of performing these digestion techniques is provided below, purelyby way of example, such description not limiting the scope of thepresent invention.

Nitrous Acid Digestion

Nitrous acid based depolymerisation of heparan sulphate leads to theeventual degradation of the carbohydrate chain into its individualdisaccharide components when taken to completion.

For example, nitrous acid may be prepared by chilling 250 μl of 0.5 MH₂SO₄ and 0.5 M Ba(NO₂)₂ separately on ice for 15 min. After cooling,the Ba(NO₂)₂ is combined with the H₂SO₄ and vortexed before beingcentrifuged to remove the barium sulphate precipitate. 125 μl of HNO₂was added to GAG samples resuspended in 20 μl of H₂O, and vortexedbefore being incubated for 15 min at 25° C. with occasional mixing.After incubation, 1 M Na₂CO₃ was added to the sample to bring it to pH6. Next, 100 μl of 0.25 M NaBH₄ in 0.1 M NaOH is added to the sample andthe mixture heated to 50° C. for 20 min. The mixture is then cooled to25° C. and acidified glacial acetic acid added to bring the sample to pH3. The mixture is then neutralised with 10 M NaOH and the volumedecreased by freeze drying. Final samples are run on a Bio-Gel P-2column to separate di- and tetrasaccharides to verify the degree ofdegradation.

Lyase Digestion

Heparinise III cleaves sugar chains at glucuronidic linkages. The seriesof Heparinase enzymes (I, II and III) each display relatively specificactivity by depolymerising certain heparan sulphate sequences atparticular sulfation recognition sites. Heparinase I cleaves HS chainswith NS regions along the HS chain. This leads to disruption of thesulphated domains. Heparinase III depolymerises HS with the NA domains,resulting in the separation of the carbohydrate chain into individualsulphated domains. Heparinase II primarily cleaves in the NA/NS“shoulder” domains of HS chains, where varying sulfation patterns arefound. Note: The repeating disaccharide backbone of the heparan polymeris a uronic acid connected to the amino sugar glucosamine. “NS” meansthe amino sugar is carrying a sulfate on the amino group enablingsulfation of other groups at C2, C6 and C3. “NA” indicates that theamino group is not sulphated and remains acetylated.

For example, for depolymerisation in the NA regions using Heparinase IIIboth enzyme and lyophilised HS samples are prepared in a buffercontaining 20 mM Tris-HCL, 0.1 mg/ml BSA and 4 mM CaCl₂ at pH 7.5.Purely by way of example, Heparinase III may be added at 5 mU per 1 μgof HS and incubated at 37° C. for 16 h before stopping the reaction byheating to 70° C. for 5 min.

Di- and tetrasaccharides may be eluted by column chromatography.

Heparin-Binding Domains

Cardin and Weintraub (Molecular Modeling of Protein-GlycosaminoglycanInteractions, Arteriosclerosis Vol. 9 No. 1 January/February 1989 p.21-32), incorporated herein in entirety by reference, describesconsensus sequences for polypeptide heparin-binding domains. Theconsensus sequence has either a stretch of di- or tri-basic residuesseparated by two or three hydropathic residues terminated by one or morebasic residues. Two particular consensus sequences were identified:XBBXBX [SEQ ID NO:3] and XBBBXXBX [SEQ ID NO:4] in which B is a basicresidue (e.g. Lysine, Arginine, Histidine) and X is a hydropathicresidue (e.g. Alanine, Glycine, Tyrosine, Serine). Heparin-bindingdomains are reported to be abundant in amino acids Asn, Ser, Ala, Gly,Ile, Leu and Tyr and have a low occurrence of amino acids Cys, Glu, Asp,Met, Phe and Trp.

These consensus sequences may be used to search protein or polypeptideamino acid sequences in order to identify candidate heparin-bindingdomain amino acid sequences which may be synthesised and tested for GAGbinding in accordance with the present invention.

WO 2005/014619 A2 also discloses numerous heparin-binding peptides. Thecontents of WO 2005/014619 A2 are incorporated herein in entirety byreference.

Bone Fracture

In some aspects the present invention is concerned with the therapeuticuse (human and veterinary) of HS/BMP2 to treat bone fracture. HS/BMP2 isreported here to augment wound healing in bone. HS/BMP2 stimulates boneregeneration following injury and contributes to improved wound healingin bone. HS/BMP2 provides improvements in the speed of bone fracturerepair enabling a reduction in the recovery time from injury.

Bone fracture is a medical condition. In this application “fracture”includes damage or injury to bone in which a bone is cracked, broken orchipped. A break refers to discontinuity in the bone. A fracture may becaused by physical impact, or mechanical stress or by medical conditionssuch as osteoporosis or osteoarthritis.

Orthopaedic classification of fractures includes closed or open andsimple or multi-fragmentary fractures. In closed fractures the skinremains intact, whilst in an open fracture the bone may be exposedthrough the wound site, which brings a higher risk of infection. Simplefractures occur along a single line, tending to divide the bone in two.Multi-fragmentary fractures spilt the bone into multiple pieces.

Other fracture types include, compression fracture, compacted fracture,spiral fracture, complete and incomplete fractures, transverse, linearand oblique fractures and comminuted fractures.

In most subjects bone healing (fracture union) occurs naturally and isinitiated following injury. Bleeding normally leads to clotting andattraction of white blood cells and fibroblasts, followed by productionof collagen fibres. This is followed by bone matrix (calciumhydroxyapatite) deposition (mineralisation) transforming the collagenmatrix into bone. Immature re-generated bone is typically weaker thanmature bone and over time the immature bone undergoes a process ofremodelling to produce mature “lamellar” bone. The complete bone healingprocess takes considerable time, typically many months.

Bones in which fractures occur and which may benefit from treatmentusing HS/BMP2 include all bone types, particularly all mammalian bonesincluding, but not limited to, long bones (e.g. femur, humerus,phalanges), short bones (e.g. carpals, tarsals), flat bones (e.g.cranium, ribs, scapula, sternum, pelvic girdle), irregular bones (e.g.vertebrae), sesamoid bones (e.g. patella).

Bones in which fractures occur and which may benefit from treatmentusing HS/BMP2 include skeletal bone (i.e. any bone of the skeleton),bones of the cranio-facial region, bones of the axial skeleton (e.g.vertebrae, ribs), appendicular bone (e.g. of the limbs), bone of thepelvic skeleton (e.g. pelvis).

Bones in which fractures occur and which may benefit from treatmentusing HS/BMP2 also include those of the head (skull) and neck, includingthose of the face such as the jaw, nose and cheek. In this respect, insome preferred embodiments HS/BMP2 may be used to assist in repair orregeneration of bone during dental or facial or cranial surgery, whichmay include reconstruction of bones (as distinct from teeth) of the faceand/or mouth, e.g. including the jawbone. In some embodiments HS/BMP2 isused for the repair or regeneration of alveolar bone. This may be repairor regeneration of dental alveolar bone repair where HS/BMP2 may beadministered during craniofacial and/or craniomaxillofacial surgery.

Bone fracture also includes pathological porosity, such as thatexhibited by subjects with osteoporosis.

Although not limiting to the present invention, the primary actions ofHS/BMP2 may be on cells within, adjacent to, or caused to migrate intothe wound site and may be on the bone stem cells, the preosteoblasts orthe osteoblasts, or on any of the ancillary or vasculogenic cells foundor caused to migrate into or within the wound bed.

HS/BMP2 and pharmaceutical compositions and medicaments comprisingHS/BMP2 are provided for use in a method of treatment of bone fracturein a mammalian subject.

Treatment may comprise wound healing in bone. The treatment may involverepair, regeneration and growth of bone. HS/BMP2 facilitates fracturerepair by facilitating new bone growth. HS/BMP2 acts to improve thespeed of fracture repair enabling bone healing to occur faster leadingto improved recovery time from injury. Treatment may lead to improvedbone strength.

Treatment may also include treatment of osteoporosis or osteoarthritis.

Administration of HS/BMP2 is preferably to the tissue surrounding thefracture. This may include administration directly to bone tissue inwhich the fracture has occurred.

Administration may be to connective tissue surrounding the bone orfracture or to vasculature (e.g. blood vessels) near to and supplyingthe bone. Administration may be directly to the site of injury and maybe to a callus formed by initial healing of the wound.

Medicaments and pharmaceutical compositions according to the presentinvention may be formulated for administration by a number of routes.Most preferably HS/BMP2 is formulated in fluid or liquid form forinjection.

In some embodiments the HS/BMP2 is formulated as a controlled releaseformulation, e.g. in a drug capsule for implantation at the wound site.The HS/BMP2 may be attached to, impregnated on or soaked into a carriermaterial (e.g. a biomaterial) such as nanofibres or biodegradable paperor textile.

Pharmaceutical compositions, medicaments, implants and prosthesescomprising

HS/BMP2 may also comprise BMP2. Owing to the ability of HS/BMP2 to bindBMP2, the HS/BMP2 may act as a carrier of BMP2 assisting in delivery ofBMP2 to the wound site and maintenance of BMP2 stability.

Administration is preferably in a “therapeutically effective amount”,this being sufficient to improve healing of the bone fracture comparedto a corresponding untreated fracture. The actual amount administered,and rate and time-course of administration, will depend on the natureand severity of the fracture. Prescription of treatment, e.g. decisionson dosage etc, is within the responsibility of general practitioners andother medical doctors, and will typically take account of the nature ofthe fracture, the condition of the individual patient, the site ofdelivery, the method of administration and other factors known topractitioners. Single or multiple administrations of HS/BMP2 doses maybe administered in accordance with the guidance of the prescribingmedical practitioner. Purely by way of example, HS/BMP2 may be deliveredin dosages of at least 1 ng/ml, more preferably at least 5 ng/ml andoptionally 10 ng/ml or more. Individual HS/BMP dosages may be of theorder less than 1 mg and greater than 1 μg, e.g. one of about 5 μg,about 10 μg, about 25 μg, about 30 μg, about 50 μg, about 100 μg, about0.5 mg, or about 1 mg. Examples of the techniques and protocolsmentioned above can be found in Remington's Pharmaceutical Sciences,20th Edition, 2000, pub. Lippincott, Williams & Wilkins.

HS/BMP2 may be used to treat bone fracture alongside other treatments,such as administration of pain relieving or anti-inflammatorymedicaments, immobilisation and setting of the bone, e.g. immobilisingthe injured limb in a plaster cast, surgical intervention, e.g. tore-set a bone or move a bone to correct displacement, angulation ordislocation. If surgery is required HS/BMP2 may be administered directlyto (e.g. applied to) the fracture during the surgical procedure.

Biomaterials

Pharmaceutical compositions and medicaments of the invention may takethe form of a biomaterial that is coated and/or impregnated withHS/BMP2. An implant or prosthesis may be formed from the biomaterial.Such implants or prostheses may be surgically implanted to assist inbone growth, regeneration, restructuring and/or re-modelling.

HS/BMP2 may be applied to implants or prostheses to accelerate new boneformation at a desired location. It will be appreciated that heparansulphates, unlike proteins, are particularly robust and have a muchbetter ability to withstand the solvents required for the manufacture ofsynthetic bioscaffolds and application to implants and prostheses.

The biomaterial may be coated or impregnated with HS/BMP2. Impregnationmay comprise forming the biomaterial by mixing HS/BMP2 with theconstitutive components of the biomaterial, e.g. during polymerisation,or absorbing HS/BMP2 into the biomaterial. Coating may compriseadsorbing the HS/BMP2 onto the surface of the biomaterial.

The biomaterial should allow the coated or impregnated HS/BMP2 to bereleased from the biomaterial when administered to or implanted in thesubject. Biomaterial release kinetics may be altered by altering thestructure, e.g. porosity, of the biomaterial.

In addition to coating or impregnating a biomaterial with HS/BMP2, oneor more biologically active molecules may be impregnated or coated onthe biomaterial. For example, at least one chosen from the groupconsisting of: BMP-2, BMP-4, OP-1, FGF-1, FGF-2, TGF-β1, TGF-β2, TGF-β3;VEGF; collagen; laminin; fibronectin; vitronectin. In addition oralternatively to the above bioactive molecules, one or morebisphosphonates may be impregnated or coated onto the biomaterial alongwith HS/BMP2. Examples of useful bisphosphonates may include at leastone chosen from the group consisting of: etidronate; clodronate;alendronate; pamidronate; risedronate; zoledronate.

Biomaterials coated or impregnated with HS/BMP2 may be useful in bothmedical and veterinary purposes. It will be appreciated that the presentinvention may improve the quality of life of a patient or potentiallyextend the life of an animal, for example a valuable racehorse for usein breeding.

The biomaterial provides a scaffold or matrix support. The biomaterialmay be suitable for implantation in tissue, or may be suitable foradministration (e.g. as microcapsules in solution).

The implant or prosthesis should be biocompatible, e.g. non-toxic and oflow immunogenicity (most preferably non-immunogenic). The biomaterialmay be biodegradable such that the biomaterial degrades as wound healingoccurs, ultimately leaving only the regenerated bone in situ in thesubject. Alternatively a non-biodegradable biomaterial may be used, e.g.to guide bone regeneration over a large discontinuity and/or to act as astructural support during bone healing, with surgical removal of thebiomaterial being an optional requirement after successful woundhealing.

Biomaterials may be soft and/or flexible, e.g. hydrogels, fibrin web ormesh, or collagen sponges. A “hydrogel” is a substance formed when anorganic polymer, which can be natural or synthetic, is set or solidifiedto create a three-dimensional open-lattice structure that entrapsmolecules of water or other solutions to form a gel. Solidification canoccur by aggregation, coagulation, hydrophobic interactions orcross-linking.

Alternatively biomaterials may be relatively rigid structures, e.g.formed from solid materials such as plastics or biologically inertmetals such as titanium.

The biomaterial may have a porous matrix structure which may be providedby a cross-linked polymer. The matrix is preferably permeable tonutrients and growth factors required for bone growth.

Matrix structures may be formed by crosslinking fibres, e.g. fibrin orcollagen, or of liquid films of sodium alginate, chitosan, or otherpolysaccharides with suitable crosslinkers, e.g. calcium salts,polyacrylic acid, heparin. Alternatively scaffolds may be formed as agel, fabricated by collagen or alginates, crosslinked using wellestablished methods known to those skilled in the art.

Suitable polymer materials for matrix formation include, but are notlimited by, biodegradable/bioresorbable polymers which may be chosenfrom the group of: agarose, collagen, fibrin, chitosan,polycaprolactone, poly(DL-lactide-co-caprolactone),poly(L-lactide-co-caprolactone-co-glycolide), polyglycolide,polylactide, polyhydroxyalcanoates, co-polymers thereof, ornon-biodegradable polymers which may be chosen from the group of:cellulose acetate; cellulose butyrate, alginate, polysulfone,polyurethane, polyacrylonitrile, sulfonated, polyamide,polyacrylonitrile, polymethylmethacrylate, co-polymers thereof.

Collagen is a promising material for matrix construction owing to itsbiocompatibility and favourable property of supporting cell attachmentand function (U.S. Pat. No. 5,019,087; Tanaka, S.; Takigawa, T.;Ichihara, S. & Nakamura, T. Mechanical properties of the bioabsorbablepolyglycolic acid-collagen nerve guide tube Polymer Engineering &Science 2006, 46, 1461-1467). Clinically acceptable collagen sponges areone example of a matrix and are well known in the art (e.g. from IntegraLife Sciences).

Fibrin scaffolds (e.g. fibrin glue) provide an alternative matrixmaterial. Fibrin glue enjoys widespread clinical application as a woundsealant, a reservoir to deliver growth factors and as an aid in theplacement and securing of biological implants (Rajesh Vasita, DhirendraS Katti. Growth factor delivery systems for tissue engineering: amaterials perspective. Expert Reviews in Medical Devices. 2006; 3(1):29-47; Wong C, Inman E, Spaethe R, Helgerson S. Thromb. Haemost. 200389(3): 573-582; Pandit A S, Wilson D J, Feldman D S. Fibrin scaffold asan effective vehicle for the delivery of acidic growth factor (FGF-1).J. Biomaterials Applications. 2000; 14(3); 229-242; DeBlois Cote M F.Doillon C J. Heparin-fibroblast growth factor fibrin complex: in vitroand in vivo applications to collagen based materials. Biomaterials.1994; 15(9): 665-672.).

Luong-Van et al (In vitro biocompatibility and bioactivity ofmicroencapsulated heparan sulphate Biomaterials 28 (2007) 2127-2136),incorporated herein by reference, describes prolonged localised deliveryof HS from polycaprolactone microcapsules.

A further example of a biomaterial is a polymer that incorporateshydroxyapatite or hyaluronic acid.

One example of a biomaterial suitable for use in combination withHS/BMP2 is the JAX™ bone void filler (Smith & Nephew). Jax granules arecomposed of high purity calcium sulfate and retain their shape toprovide a scaffold with controlled, inter-granular porosity and granulemigration stability. Jax granules dissolve safely and completely in thebody.

Other suitable biomaterials include ceramic or metal (e.g. titanium),hydroxyapatite, tricalcium phosphate, demineralised bone matrix (DBM),autografts (i.e. grafts derived from the patient's tissue), orallografts (grafts derived from the tissue of an animal that is not thepatient). Biomaterials may be synthetic (e.g. metal, fibrin, ceramic) orbiological (e.g. carrier materials made from animal tissue, e.g.non-human mammals (e.g. cow, pig), or human).

The biomaterial can be supplemented with additional cells. For example,one can “seed” the biomaterial (or co-synthesise it) withundifferentiated bone precursor cells, e.g. stem cells such asmesenchymal stem cells, more preferably human mesenchymal stem cells.

The subject to be treated may be any animal or human. The subject ispreferably mammalian, more preferably human. The subject may be anon-human mammal (e.g. rabbit, guinea pig, rat, mouse or other rodent(including cells from any animal in the order Rodentia), cat, dog, pig,sheep, goat, cattle (including cows, e.g. dairy cows, or any animal inthe order Bos), horse (including any animal in the order Equidae),donkey, and non-human primate). The non-human mammal may be a domesticpet, or animal kept for commercial purposes, e.g. a race horse, orfarming livestock such as pigs, sheep or cattle. The subject may be maleor female. The subject may be a patient.

Culture Media

Culture media comprising HS/BMP2 may be of any kind but is preferablyliquid or gel and may contain other nutrients and growth factors (e.g.FGF-2). HS/BMP2 will preferably be present in non-trace amounts. Forexample, the concentration of HS/BMP2 in the culture media may rangebetween about 1.0 ng/ml culture media to about 1000 ng/ml culture media.Preferably, the concentration of HS/BMP2 in the culture media is betweenabout 5 ng/ml culture media and 200 ng/ml culture media, more preferablybetween about 20 ng/ml culture media and 170 ng/ml culture media

BMP2 Protein

In this specification BMP2 refers to Bone morphogenetic protein 2 (alsocalled bone morphogenic protein 2, BMP2 or BMP-2), which is a member ofthe TGF-β superfamily and is implicated in the development of bone andcartilage.

The amino acid sequence of BMP2 preprotein from Homo sapiens (SEQ IDNO:2) is shown in FIG. 35. Amino acids 1 to 23 represent the signalpeptide, and amino acids 24 to 396 represent the amino acid sequence ofthe proprotein. The amino acid sequence of the mature protein is givenas SEQ ID NO:5 herein.

In this specification “BMP2 protein” includes proteins having at least70%, more preferably one of 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%or 100% sequence identity with the amino acid sequence of the BMP2preprotein or BMP2 proprotein illustrated in FIG. 35 or with the aminoacid sequence of the mature BMP2 protein of SEQ ID NO:5.

The BMP2 protein preferably also includes a heparin binding domainhaving the amino acid sequence of SEQ ID NO:1 or 6 (found at amino acids283-300 of SEQ ID NO:2), or an amino acid sequence having one of 75%,80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ IDNO:1 or 6.

Reference to BMP2 protein preferably includes the BMP-2 proteindescribed in Ruppert et al (Eur J. Biochem 1996).

The BMP2 protein is preferably osteogenic, i.e. has the activity ofinducing, or assisting in the induction of, osteoblast differentiation.

The BMP2 protein may be from, or derived from, any animal or human, e.g.non-human animals, e.g. rabbit, guinea pig, rat, mouse or other rodent(including from any animal in the order Rodentia), cat, dog, pig, sheep,goat, cattle (including cows, e.g. dairy cows, or any animal in theorder Bos), horse (including any animal in the order Equidae), donkey,and non-human primate or other non-human vertebrate organism; and/ornon-human mammalian animal; and/or human.

The invention includes the combination of the aspects and preferredfeatures described except where such a combination is clearlyimpermissible or expressly avoided.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.

Aspects and embodiments of the present invention will now beillustrated, by way of example, with reference to the accompanyingfigures. Further aspects and embodiments will be apparent to thoseskilled in the art. All documents mentioned in this text areincorporated herein by reference.

BRIEF DESCRIPTION OF THE FIGURES

Embodiments and experiments illustrating the principles of the inventionwill now be discussed with reference to the accompanying figures inwhich:

FIG. 1. Anion exchange chromatography of MX samples disrupted using 8 MUrea/CHAPS buffer. A large GAG peak is observed after 1M NaCl elution.

FIG. 2. Representative chromatogram of the desalting system duringMX-derived GAG purification. The initial peak (12-18 min) representsfull length GAG chains. The conductivity peak and debris peak (19-30min) represent salt and GAG debris elution.

FIG. 3. tGAGs (2.5 mg) loaded onto an underivatised Hi-Trap streptavidincolumn. All GAGs elute from the column in the flowthrough, indicating no“background” attachment of GAGs to the column.

FIG. 4. BMP2-HBP (1 mg) pre-incubated with tGAGs (25 mg) for 30 min.Elution profile shows the peptide (280 nm) exiting the column in theflowthrough together with the tGAG sample.

FIG. 5. BMP2-HBP (1 mg) loaded onto a Hi-Trap column. The 280 nmabsorbance levels indicate that the peptide remains attached to thecolumn even under high salt conditions; thus there was successfulcoupling of the biotinylated peptide to the streptavidin linker.

FIG. 6. BMP2-HBP (1 mg) coupled column loaded with of 25 mg of tGAGs.The chromatogram (232 nm) clearly shows both an overloading of thecolumn, in the flow through as well as the binding of some GAGs to theBMP2-HBP bed.

FIG. 7. Re-application of the GAG− (flowthrough) fractions from theprevious experiment (FIG. 6). The presence of a significant GAG+ elutionpeak indicates that all available BMP2-HBP binding sites had beensaturated, resulting in a large proportion of susceptible GAGs exitingthe column in the flowthrough.

FIG. 8. BMP2-HBP (2 mg) coupled column loaded with tGAGs (6 mg). Thechromatogram (232 nm) clearly shows no overloading of the column, andthe presence of a GAG subpopulation with a relative affinity for theBMP2-HBP.

FIG. 9. Re-run of GAG− (flowthrough) from previous run (FIG. 8). Theabsence of a GAG+ elution peak indicates that the available BMP2-HBPbinding sites were not saturated in the previous run, allowing theefficient extraction of GAG+ sugars in a single run.

FIG. 10. Re-application of isolated full length GAG+ fractions (2 mg)shows no change in affinity for the BMP2-HBP (2 mg) column prior toheparinase III digestion. A reapplication of GAG− fractions against theBMP2-HBP column also showed no change in affinity, with all GAGs exitingthe column in the flowthrough essentially as in FIG. 9.

FIG. 11. GAG− fractions (1 mg) digested with heparinase III beforeloading onto the BMP2-HBP (2 mg) column. The chromatogram (232 nm)clearly shows that no GAG samples remain bound to the column, but exitin the flowthrough. This indicates the absence of any GAG+ domains inthe full length GAG− chains.

FIG. 12. GAG+ fractions (2 mg) digested with heparinase 3 before loadingonto the BMP2-HBP (2 mg) column. The chromatogram (232 nm) demonstratesthat the GAG+ samples are retained by the column, suggesting that alldomains on the full length GAG+ chain have a relative affinity for theBMP2-HBP. The increase in the absorbance peak, as compared to the samedry weight quantity of GAG+ (FIG. 10), indicates the efficacy of theheparinase 3 treatment.

FIG. 13. Full length GAG+ chains separated using a Biogel P10 columnwith an exclusion limit of between 1.5 kDa and 20 kDa. The chromatogramshows that a large proportion of the sample chains have an overallmolecular weight of more than 20 kDa.

FIG. 14. Full length GAG+ sugar chains treated with nitrous acid for 20min to diagnostically degrade heparan sulfate species. The chromatogram,generated from a Biogel P10 sizing column, shows an almost completedegradation of all GAG+ chains as compared to FIG. 13, indicating thatGAG+ isolated chains consist primarily of heparan sulfate.

FIG. 15. Chondroitin-4-sulfate (6 mg) loaded onto BMP2-HBP (2 mg)column. The chromatogram clearly illustrates a significant proportion ofthe GAG chains having an affinity for the peptide, as they eluted at asimilar salt concentration as the GAG+ samples.

FIG. 16. Chondroitin-6-sulfate (6 mg) loaded onto BMP2-HBP (2 mg)column. The chromatogram indicates that few of the C6S GAG chains haveany affinity for the peptide column.

FIG. 17. Dermatan sulfate (6 mg) loaded onto the BMP2-HBP (2 mg)affinity column. The chromatogram indicates that few of the DS GAGchains had any affinity for the peptide, with only a small proportion ofthe GAGs being eluted at a similar salt concentration to GAG+ samples.

FIG. 18. Bovine heparan sulfate (2.5 mg) loaded onto the BMP2-HBP (2 mg)column. The chromatogram (232 nm) reveals only a small fraction of theGAGs binding to the column.

FIG. 19. Heparin-LMW (50 mg) loaded onto the BMP2-HBP (2 mg) column. Thechromatogram (232 nm) reveals that almost no GAG bound to the peptide.

FIG. 20. Heparin-HMW (28 mg) loaded onto the BMP2-HBP (2 mg) column. Thechromatogram (232 nm) reveals that almost no GAG bound to the peptide.

FIG. 21. Heparin-HMW (25 mg) predigested with heparinase I was loadedonto the BMP2-HBP (2 mg) column. The chromatogram (232 nm) reveals thatvery few GAG fragments bound to the peptide.

FIG. 22. Chromatogram showing steps in isolation of BMP-2 peptidespecific HS by affinity chromatography.

FIG. 23. Chromatogram showing elution of BMP-2 peptide specific HS(GAG+) by affinity chromatography.

FIG. 24. Chromatogram showing elution of BMP-2 peptide non-specific HS(GAG−) by affinity chromatography.

FIG. 25. Chromatogram showing elution of Sigma HS(H9902) standard undersize exclusion chromatography on Superdex 75 column.

FIG. 26. Chromatogram showing elution of BMP-2 peptide specific HS(GAG+) under size exclusion chromatography on Superdex 75 column.

FIG. 27. Graph showing Osterix expression in C2C12 cells in response tocontrol media, 100 ng/ml BMP2 and 300 ng/ml BMP2.

FIG. 28. Graph showing Osteocalcin expression in C2C12 cells in responseto control media, 100 ng/ml BMP2 and 300 ng/ml BMP2.

FIG. 29. Graph showing Runx2 expression in C2C12 cells in response tocontrol media, 100 ng/ml BMP2 and 300 ng/ml BMP2.

FIG. 30. Graph showing expression of Alkaline Phosphatase as measured byquantative PCR in C2C12 cells in response to control media, BMP-2,Negative GAG (GAG−), Positive GAGs (GAG+), Total HS and Heparin (Hep).

FIG. 31. Graph showing expression of Osterix as measured by quantativePCR in C2C12 cells in response to control media, BMP-2, Negative GAG(GAG−)+BMP-2, Positive GAGs (GAG+)+BMP-2, Total HS and Heparin (Hep).

FIG. 32. Graph showing expression of BspII as measured by quantative PCRin C2C12 cells in response to control media, BMP-2, Negative GAG(GAG−)+BMP-2, Positive GAGs (GAG+)+BMP-2, Total HS and Heparin (Hep).

FIG. 33. Graph showing expression of Runx2 as measured by quantative PCRin C2C12 cells in response to control media, BMP-2, Negative GAG(GAG−)+BMP-2, Positive GAGs (GAG+)+BMP-2, Total HS and Heparin (Hep).

FIG. 34. Graph showing expression of Osteocalcin in C2C12 cells inresponse to BMP and GAG+ (+BMP-2) isolated from MC3T3-E1 cells.

FIG. 35. Amino acid sequence of bone morphogenetic protein 2 preproteinfrom Homo sapiens, NCBI Accession No. NP_(—)001191 (NP_(—)001191.1GI:4557369) (SEQ ID NO:2).

FIG. 36. Chromatogram showing elution of BMP-2 peptide specific HS byaffinity chromatography. 6 mg Biotinylated BMP2-peptide (SEQ ID NO:1)was coupled to a 1 ml Streptavidin column. The chromatogram shows thatall of the biotinylated BMP2-peptide bound to the column.

FIG. 37. Chromatogram showing purification of BMP2-peptide (SEQ ID NO:1)specific heparan sulphate.

FIG. 38. Chromatogram showing desalting of BMP2 peptide (SEQ ID NO:1)column bound heparan sulphate.

FIG. 39. Chromatogram showing desalting of BMP2 peptide (SEQ ID NO:1)column unbound heparan sulphate.

FIG. 40. SAX-HPLC profile following disaccharide digestion of BMP2positive heparan sulphate.

FIG. 41. SAX-HPLC profile following disaccharide digestion of BMP2negative heparan sulphate.

FIG. 42. SAX-HPLC profile following disaccharide digestion of Celsus HS.

FIG. 43. Table showing lyase-derived disaccharide percentage compositionof BMP2-specific HS, BMP2-non-specific HS and Celsus HS. The area undereach peak was integrated to calculate the percentage of eachdisaccharide.—=not detected.

FIG. 44. Chart showing surface plasmon resonance (SPR) analysis of BMP2positive and BMP2 negative HS.

FIG. 45. Chart showing BMP2 binding capacity of BMP2 positive and BMP2negative Celsus HS preparations coated on an Iduron Heparin/GAG bindingplate.

FIG. 46. Chart showing Alkaline Phosphatase (ALP) activity of BMP2positive and BMP2 negative HS on C2C12 cells.

FIG. 47. Photographs of immunohistochemical analysis of HS enhancementof ALP activity. BMP2 specific HS enhanced ALP activity induced by BMP2at a greater degree compared to non-specific HS when evaluatedhistochemically. BMP2 at 100 ng/ml was introduced in combination with 0,0.3, 3 and 30 μg/ml of BMP2 positive or BMP2 negative HS.

FIG. 48. No Figure.

FIG. 49. Chart showing BMP2 binding capacity of selectively (2-O, 6-Oand N—) de-sulfated BMP2 positive HS and indicating charge-substitutionpattern of HS chains required for binding to BMP2.

FIG. 50. Chart showing effect of heparin on BMP-2 stability.

FIG. 51. SEM photograph of JAX™—tricalcium phosphate bone filler, X-rayphotographs of Rabbit ulna defect model. Illustration of combinationwith 30 μg HS/BMP2 in 200 μl hydrogel (88% water, glycerol, sodiumcarboxymethyl cellulose).

FIG. 52. X-ray and microCT scan analysis of Rabbit ulna defect modeltreated with JAX™ bone filler (control) or JAX™ bone filler plus HS/BMP2at 4 weeks from treatment.

FIG. 53. X-ray and microCT scan analysis of Rabbit ulna defect modeltreated with JAX™ bone filler (control) or JAX™ bone filler plus HS/BMP2at 8 weeks from treatment.

FIG. 54. Charts showing % bone volume as assessed by microCT scan inRabbit ulna defect model treated with JAX™ bone filler (control), JAX™bone filler plus HS/BMP2 (HS3) or JAX™ bone filler plus BMP2 negative HSat (A) 4 weeks from treatment and (B) 8 weeks from treatment.

FIG. 55. Immunoblot showing levels of Smad 1/5/8 phosphorylationfollowing exposure to negative control, BMP2 alone, BMP2+Heparin orBMP2+HS3.

FIG. 56. Diagrammatic illustration of experimental design of non-unioncritical rabbit ulna defect repair.

FIG. 57. Chart showing percentage release of heparin from JAX™ granulesover time.

FIG. 58. X-ray micrographs showing healing rabbit ulna defect modeltreated with Jax™ bone filler plus control (no HS), 30 μg HS3 (HS30) or100 μg HS3 (HS100) at weeks 0, 4 and 8.

FIG. 59. Micrographs showing micro CT (computerized tomography) with 3Dimage rendering of the Jax stars within bone defects after 4 and 8 weekspost-surgery, compared to image-processed X-ray reconstructions (newbone in yellow). MicroCT rendered images in grey to the right of theX-ray images.

FIG. 60. Chart showing quantification of the % bone volume of totalvolume (BV/TV) for the treatment groups (control Vs HS30 and HS100) atweeks 4 and 8.

FIG. 61. Micrographs showing H&E staining (vide infra) for the 3treatment groups (control (no HS), 30 μg HS3 (HS30) or 100 μg HS3(HS100)) over weeks 4 and 8.

FIG. 62. Higher magnification micrographs (compared with FIG. 61)showing H&E staining for the 3 treatment groups (control (no HS), 30 μgHS3 (HS30) or 100 μg HS3 (HS100)) over weeks 4 and 8.

FIG. 63. Micrographs showing Ralis Tetrachrome staining (vide infra) forthe 3 treatment groups (control (no HS), 30 μg HS3 (HS30) or 100 μg HS3(HS100)) over weeks 4 and 8.

FIG. 64. Higher magnification micrographs (compared with FIG. 63)showing Ralis Tetrachrome staining for the 3 treatment groups (control(no HS), 30 μg HS3 (HS30) or 100 μg HS3 (HS100)) over weeks 4 and 8.

FIG. 65. Micrographs showing osteocalcin immunostaining (vide infra) forthe 3 treatment groups (control (no HS), 30 μg HS3 (HS30) or 100 μg HS3(HS100)) over weeks 4 and 8.

FIG. 66. Higher magnification micrographs (compared with FIG. 65)showing osteocalcin immunostaining for the 3 treatment groups (control(no HS), 30 μg HS3

(HS30) or 100 μg HS3 (HS100)) over weeks 4 and 8.

FIG. 67. Photographic illustration of torsional testing apparatus.

FIG. 68. Charts showing typical torsion vs. angle for intact ulna, plusstiffness and maximum torque for HS30 at week 8.

FIG. 69. X-ray micrographs at 0 weeks showing rabbit ulna defect modeltreated with collagen sponges soaked with one of the followingtreatments (total 300 μL, in PBS): 30 μg HS3, 10 μg BMP-2, 30 μg HS3+10μg BMP-2 or an equal volume of PBS.

FIG. 70. X-ray micrographs at 4 weeks showing healing in rabbit ulnadefect model treated with collagen sponges soaked with one of thefollowing treatments (total 300 μL, in PBS): 30 μg HS3, 10 μg BMP-2, 30μg HS3+10 μg BMP-2 or an equal volume of PBS.

FIG. 71. X-ray micrographs at 8 weeks showing healing in rabbit ulnadefect model treated with collagen sponges soaked with one of thefollowing treatments (total 300 μL, in PBS): 30 μg HS3, 10 μg BMP-2, 30μg HS3+10 μg BMP-2 or an equal volume of PBS.

FIG. 72. Chart showing micro CT analysis at 4 and 8 weeks for rabbitulna defect model treated with collagen sponges soaked with one of thefollowing treatments (total 300 μL, in PBS): 30 μg HS3, 10 μg BMP-2, 30μg HS3+10 μg BMP-2 or an equal volume of PBS.

FIG. 73. Charts showing maximum torque and stiffness at week 8 forrabbit ulna defect model treated with collagen sponges soaked with oneof the following treatments (total 300 μL, in PBS): 30 μg HS3, 10 μgBMP-2, 30 μg HS3+10 μg BMP-2 or an equal volume of PBS.

FIG. 74. Chart and micro CT image showing percentage bone volume at week4 for rabbit ulna defect model treated with collagen sponges soaked withone of the following treatments (total 300 μL, in PBS): 30 μg HS3, 10 μgBMP-2, 30 μg HS3+10 μg BMP-2 or an equal volume of PBS.

FIG. 75. Charts showing comparison at week 4 of percentage bone volumein the rabbit ulna defect model between treatment with Jax™ TCP starsand collagen sponges when combined with one of 10 μg BMP-2 (BMP10), 30μg HS3 (HS30), 10 μg BMP-2+30 μg HS3, 100 μg HS3 (HS100) or control.

FIG. 76. Table showing disaccharide composition of Celsus HS, HSnon-BMP2 binding fraction (Not retained) and three HS/BMP2 isolates(848/HS3/001, HS3-001-01, and HS3-001-02) after capillaryelectrophoresis analysis of disaccharides in heparin lyase I, II and IIdigests of the respective heparan sulphates.

FIG. 77. Graph showing capillary electrophoresis analysis ofdisaccharides in heparin lyase I, II and III digests of heparan sulphatepreparations (Celsus HS, BMP2 Not retained and retained (HS/BMP2)),including appropriate confidence intervals (Error intervals weredetermined using student's t-distribution with confidence limits set at95). (1=ΔUA,2S-GlcNS,6S, 2=ΔUA,2S-GlcNS, 3=ΔUA-GlcNS,6S,4=ΔUA,2S-GlcNAc,6S, 5=ΔUA-GlcNS, 6=ΔUA,2S-GlcNAc, 7=ΔUA-GlcNAc,6S,8=ΔUA-GlcNAc)

FIG. 78. Graph showing capillary electrophoresis analysis ofdisaccharides in heparin lyase I, II and Ill digests of heparan sulphatepreparations (all three are BMP2 retained preparations, i.e. HS/BMP2isolates), including appropriate confidence intervals (Error intervalswere determined using student's t-distribution with confidence limitsset at 95). (1=ΔUA,2S-GlcNS,6S, 2=ΔUA,2S-GlcNS, 3=ΔUA-GlcNS,6S,4=ΔUA,2S-GlcNAc,6S, 5=ΔUA-GlcNS, 6=ΔUA,2S-GlcNAc, 7=ΔUA-GlcNAc,6S,8=ΔUA-GlcNAc)

FIG. 79. Table showing dissacharide composition of Porcine Mucosal HS(Celsus HS), BMP2 HS−ve and BMP2 HS+ve. Normalised composition of HS asdetermined by enzymatic depolymerization and capillary electrophoresisanalysis. Mean of duplicate analyses of duplicate hydrolysates witherror intervals representing student's t-distribution (95% confidence).

FIG. 80. BMP-2 binding to HS+ve. (a) Graph showing BMP-2 binding abilityof HSpm, purified HS+ve, and HS−ve coated on an Iduron Heparin/GAGbinding plate, (b) Dot blot analysis of HSpm, HS+ve and HS−ve binding toBMP-2. (c-e) Comparative binding ability of different growth factors(BMP-2, FGF2, VEGF and PDGF) with HSpm or HS+ve or HS−ve.

FIG. 81. Graphs showing BMP-2 binding to GAGs. HSpm, HS+ve and HS−ve.(A-C) BMP-2 binding ability of different concentrations of HSpm, HS+veand HS−ve coated on an Iduron heparin/GAG binding plate.

FIG. 82. Graphs showing results of Biacore binding analysis. Theincubation of HSpm (A), HS+ve (B) and HS−ve (C) with differentconcentrations of BMP-2, BMP-4 and BMP-7.

FIG. 83. BMP-2 localization on the surface of C2C12 cells. (a) Chartshowing BMP-2 binding to the cell surface as confirmed in C2C12 cells.(b) Chart showing that the addition of exogenous GAGs (HSpm, HS+ve andHS−ve) reduced the amount of BMP-2 bound on the surface of C2C12 cellsdose-dependently.

FIG. 84. HS+ve prolonged BMP-2 stability and activity. (a) Graph showingBMP-2 stability over time measured by incubating 100 ng/ml of BMP-2 inthe presence or absence of 5 μg/ml of HSpm or HS+ve or HS−ve for up to72 h. (b) Gel micrograph showing results of incubating BMP-2 with HSpmor HS+ve or HS−ve for up to 72 h to test for its biologically activityas indicated by its ability to induced Smad 1/5/8 phosphorylation(p-Smad 1/5/8) in C2C12 cells. The cells were lysed at the indicatedtime points, and phosphorylated Smad 1/5/8 and total Smad 1/5/8 proteinswere determined by Western blotting. (C) Gel micrograph: BMP-2 (100ng/mL) was coimmunoprecipitated with 640 ng/mL BMPR-IA/Fc or BMPR-II inthe presence/absence of 5 μg/mL HSpm or HS+ve or HS−ve and separated ona Western blot.

FIG. 85. HS+ve modulate osteogenic activity of BMP-2. (a) Graph showingeffect of HSpm or HS+ve or HS−ve on BMP-2-induced ALP activity. C2C12cells were seeded in medium with 10% FCS overnight, changed to mediumwith 5% FCS containing BMP-2 with or without heparin or HS+ve or HS−ve,or HSpm and cultured for 3 days, and ALP activity determined. Theresults are the means of three independent determinations ±SD. *)significant increase in ALP activity compared to BMP-2 treatment alone(p<0.001). **) significant increase in ALP activity compared to BMP-2and HS(pm) treatment combination (p=0.004). (b) Photograph showing ALPstaining: C2C12 cells were fixed and stained for ALP. (c) Graph showingeffect of HSpm, HS+ve and HS−ve on the inhibitory effect of noggintoward BMP-2 activity through disrupting their interaction. BMP-2 at 100ng/ml was pre-incubated with or without 5 μg/ml of GAG, and introducedto C2C12 cells for 3 days together with noggin at the indicatedconcentration. Values were normalized to ALP activity observed in cellstreated with BMP-2 in combination with each respective GAG in theabsence of noggin. *) significantly lower ALP activity compared to eachGAG's respective control (i.e. GAG and BMP-2 without Noggin) (p<0.001).**) significantly lower ALP activity compared to BMP-2 treatment incombination with the corresponding noggin concentration and in theabsence of GAG (p<0.001). #) significantly higher ALP activity comparedto BMP-2 treatment in combination with the corresponding nogginconcentration and in the absence of GAG (p=<0.001).

FIG. 86. HS+ve enhances BMP-2 induced mineralization. Photograph showingresult of culturing C2C12 cells in osteogenic media in the presence orabsence of 100 ng/mL BMP-2 and 5 μg/mL of HSpm or HS+ve or HS−ve for 14days followed by Alizarin Red staining.

FIG. 87. HS+ve has minimal anti-coagulant activity compared to heparin.At the indicated concentration, GAG was incubated with antithrombin (AT)prior to the addition of factor Xa (FXa). Any FXa that was not inhibitedby AT was measured photometrically using chromogenic substrate S-2222.The graph shows relative inhibition measured by normalizing the valuesto that of the treatment group containing no GAG.

FIG. 88. Graph showing results of Luciferase reporter assays. (a) Id1promoter luciferase reporter assay. (b) RUNX2 transcription activityassay.

FIG. 89. Graph showing HS+ve does not stimulate tumor necrosisfactor-alpha production in macrophages in vitro after 24 h. LPS:Lipopolysaccharide (positive control), Unstimulated: No added factors(negative control), HS+ve alone: 5 μg of HS exogenously added, heparin:5 μg of heparin exogenously added, Ctrl: media from plain collagensponges, HS+ve: conditioned media from collagen sponges loaded with 5 μgof HS+ve. Results are expressed as mean±standard deviation, n=3 for eachtreatment.

FIG. 90. 2 treatments were implanted on each side of a mandible (A) andallowed to recover for up to 6 months. Representative photograph (B) andX-ray (C) at sacrifice.

FIG. 91. At both 3 and 6 months post-implantation, p-CT images (A)showed more new bone formation in both the 10 μg BMP-2 and 30 μg HS3treatments than scaffold alone. (B) Notably, 30 μg HS3 was able to formas much alveolar bone as 10 μg BMP-2. (C) Chart showing BV/TV %—resultsare presented as mean±standard error of mean and significance is takenas p<0.05.

FIG. 92. Representative toluidine blue/McNeal-stained sections revealthe infiltration of new bone (b) into the defects for both treatments tobe mainly from the lower host alveolar bone, with some infiltrating fromthe sides. This was evident at both 3 (A) and 6 months (B).

FIG. 93. (A) Sequential standard 2D x-rays and 3D p-CT scans showingtransverse view of treated defects at 12 weeks. * New bone filling upthe defect was evident in the 5 μg BMP-2 and 30 μg HS3+ve-treatedcalvarial defects. (B) Histological Sections Showing Cross-section ofEntire Treated Defects at 12 weeks. Dotted rectangle: Defect area, NB:New Bone. (C) Graph showing Extent of Mineralization Measured by p-CT at12 Weeks Results are presented as mean±STD, N=6/group, *p<0.05. (D)Photographs showing retention of BMP-2 and HS3+ve in a treated rat invivo.

FIG. 94. (A) Graph showing HS3 binding to BMP-2, FGF2, VEGF, PDGFB. (B)Graph showing effect of HS3 and BMP-2 on induction of ALP activity inC2C12 cells. (C) Photograph of gels showing BMP-2-induction of p-Smad1/5/8 activity in C2C12 cells with or without HS3 at 24, 48 and 72hours.

FIG. 95. X-ray, 3D p-CT images of critical-sized ulna defects in rabbitstreated with collagen sponge alone (ctrl), 10 μg BMP-2 or 30 μg HS3 for8 weeks and hematoxylin & eosin staining micrographs showing theinfiltration of new bone from the radial interface and host bone at theosteotomized ends. b: bone, bm: bone marrow, ** positive stain, ob:osteoblasts, 100 μm.

FIG. 96. (A) Graph showing the extent of mineralization ofcritical-sized ulna defects in rabbits treated with collagen spongealone (ctrl), 10 μg BMP-2 or 30 μg HS3 for 8 weeks as measured by μ-CTat 8 weeks. (B) Table showing results of torsional testing of treateddefects at 8 weeks. Data is presented as means±STD, n=3/group, * p<0.05.

DETAILED DESCRIPTION OF THE INVENTION

The details of one or more embodiments of the invention are set forth inthe accompanying description below including specific details of thebest mode contemplated by the inventors for carrying out the invention,by way of example. It will be apparent to one skilled in the art thatthe present invention may be practiced without limitation to thesespecific details.

We investigated the potential of GAGs to augment the activities of bonemorphogenic protein 2 (BMP2). The highly osteoinductive activity of BMP2for the murine myogenic cell line C2C12 have been well characterised.Studies both in this cell line, and in vivo, have implicated a role forglycosaminoglycans in modulating this activity.

BMP2's affinity for heparin has similarly been well characterised.Numerous studies have been conducted that have sought to examine thedynamic interaction between BMP2 and GAGs. Some have proposed that theinteraction is inhibitory, and so responsible for either sequesteringthe cytokine away from the receptor or inducing its association with itsnumerous inhibitors, such as noggin, that have been shown, similarly, tohave an affinity for heparin. Alternative findings implicate theinteraction between BMP2 and GAGs is one of maintaining a localconcentration of the cytokine around cells that require its signallingin order to differentiate into the osteoblast lineage.

These findings also suggest that the association serves to significantlylengthen the half-life of the homodimer, so allowing it to remain activein the ECM for longer periods. As is the case with most systems, theactual role of this interaction is likely to be blend of some, or all ofthe above.

Although many studies have provided evidence for the interaction thatBMP2 has with model sugars, the specific interaction between the BMP2heparin-binding peptide (BMP2-HBP), a string of amino acids(QAKHKQRKRLKSSCKRHP [SEQ ID NO: 1]) located at the N-terminal end ofeach BMP2 monomer, and appropriate glycosaminoglycans has receivedrelatively little attention. A major question that arises is whetherthere is a complementary saccharide sequence embedded within an HS chainthat controls the association with an absolute, or at least relative,specificity.

We sought to isolate a sequence-specific glycosaminoglycan that couldmodulate BMP2 activity via a direct interaction with the cytokine.

Example 1 Materials and Methods Buffer Preparation

Preparation of all buffers for GAG extraction and analysis is conductedwith strict attention paid to quality. It is vital that the pH ofbuffers is maintained at the correct level and that all buffers befiltered and degassed in order to prevent the clogging of columns withprecipitates or bubbles. The formation of bubbles, in particular, cancause serious damage to columns, and in the case of sealed,pre-fabricated columns, leads to them becoming unusable.

All buffers used were filtered with 1×PBS without Ca²⁺ or Mg²⁺ (150 mMNaCl), or double distilled (ddH₂O) to make the final solutions.

Disruption Buffer

The 8M Urea/CHAPS disruption buffer consisted of PBS (150 mM NaCl) with1% CHAPS, 8M Urea and 0.02% NaN₃ to prevent contamination by microbialgrowth during storage. This solution was used to disrupt matrix (MX)samples, so was not degassed or filtered.

PGAG Anion Exchange Low Salt (250 mM) Buffer

Low salt PGAG anion exchange buffer comprised PBS (150 mM NaCl) with anadditional 100 mM NaCl. The buffer was equilibrated to pH 7.3 with NaOHand 0.02% NaN₃. The solution was then degassed under negative pressureand constant stirring until no further bubbles were released beforebeing filtered through a 0.4 μm filter.

PGAG Anion Exchange High Salt (1M) Buffer

High salt PGAG anion exchange buffer comprised PBS (150 mM NaCl) with anadditional 850 mM NaCl. The buffer was equilibrated to pH 7.3 with NaOHand 0.02% NaN₃ added. The solution was then degassed under negativepressure and constant stirring before being filtered through a 0.4 μmfilter.

Pronase/Neuraminidase PGAG Reconstitution Buffer

This buffer was used to reconstitute desalted PGAG samples after anionexchange in order to prepare them for enzymatic digestion of theassociated core proteins. It consisted of 25 mM sodium acetate(CH₃COOHNa). The buffer was equilibrated to pH 5.0 with glacial aceticacid (CH₃COOH). Both pronase and neuraminidase enzymes werereconstituted according to the manufacturer's instructions.

GAG Affinity Chromatography Low Salt (150 mM) Buffer

Low salt GAG anion exchange buffer was made using PBS (150 mM NaCl)without any additional salt. The buffer was equilibrated to pH 7.3 withNaOH and 0.02% NaN₃. The solution was degassed under negative pressureand constant stirring until no further bubbles were released beforebeing filtered through a 0.4 μm filter.

GAG Affinity Chromatography High Salt (1M) Buffer

High salt GAG anion exchange buffer was made using PBS (150 mM NaCl)with an additional 850 mM NaCl. The buffer was equilibrated to pH 7.3with NaOH and 0.02% NaN₃ was added, the solution was then degassed andfiltered through a 0.4 μm filter.

Desalting Solution

The desalting solution was made using ddH₂O that was equilibrated to pH7.0 with 0.02% NaN₃. The solution was then degassed and filtered.

Sample Preparation

Matrix samples were disrupted using Disruption Buffer (8M Urea/CHAPS),then scraped off the culture surface in this buffer and stirredovernight at 37° C. to ensure maximal lysis. The samples were thencentrifuged at 5000 g for 30 min and the supernatant was clarifiedthrough a 0.4 μm filter in preparation for PGAG extraction via anionexchange chromatography.

Column Preparation & Usage

The choice and preparation of the types of columns to be used for eachsequential step in the isolation and characterisation of GAGs is ofmajor importance for the success of the protocol. It was vital that ateach step the columns were equilibrated and cleaned with great care.

Anion Exchange Columns

Due to the relatively large quantities of MX substrate used for GAGextraction, and the high load this places on the column system, it wasnecessary to pack and prepare a large anion exchange column manually,specifically for this study. Capto Q anion exchange beads (Pharmacia)were packed into a Pharmacia XK 26 column (Pharmacia) to produce acolumn with a maximum loading capacity of 500 ml of MX lysate per run.

Prior to use, both the column and all buffers were equilibrated to roomtemperature for 30 min, before washing and equilibrating the column inPGAG Anion Exchange Low Salt (250 mM) Buffer for 30 min until allabsorbance channels remained stable. The clarified cell lysate was thenpassed through the column which was again rinsed in 500 ml of low saltbuffer to remove any nonspecifically bound debris. PGAGs were theneluted using 250 ml of PGAG Anion Exchange High Salt (1M) Buffer andlyophilised prior to desalting. The column was then rinsed in low saltbuffer and returned to 4° C. for storage.

Desalting Protocol

After PGAG/GAG isolation it was necessary to remove the high amount ofsalt that accumulated in the sample during elution from the column. Forthis step, all eluted samples of the same experimental group werecombined and loaded onto 4× Pharmacia HiPrep™ 26/10 desalting columns.Prior to use, both the columns and all solutions were equilibrated toroom temperature for 30 min before washing and equilibrating the columnin Desalting Solution for 30 min until all absorbance channels achievedstability. Lyophilised samples were reconstituted in Desalting Solutionin the minimum possible volume that resulted in a clear solution. Thiscombination of columns permitted the loading of up to 60 ml of sample.Those fractions eluting from the column first were lyophilised andretained for further separation or cell culture application. The columnswere then rinsed in Desalting Solution and returned to 4° C. forstorage.

BMP2-HBP Column Preparation

The isolation of GAGs carrying relative affinities for BMP2 wasconducted using a BMP2-HBP column. Approximately 2 mg of biotinylatedBMP2-HBP was prepared in 1 ml of the GAG Affinity Chromatography LowSalt (150 mM) Buffer. This amount was loaded onto a HiTrap StreptavidinHP column (Pharmacia) and allowed to attach to the column for 5 min. Thecolumn was then subjected to a complete run cycle in the absence ofGAGs. The column was washed in 13 ml of low salt buffer at a flow rateof 0.5 ml/min before being subjected to 10 ml of GAG High salt buffer at1 ml/min. Finally the column was rinsed with 10 ml of low salt buffer.During this process data was carefully monitored to ensure that nopeptide elution or column degradation was observed.

GAG+ Sample Isolation

Once the BMP2-HBP column had been prepared and tested for stabilityunder normal running conditions, it was ready to be used for theseparation of GAG+ chains from tGAG (total GAG) samples. tGAG samples (6mg) were prepared in 3 ml of GAG affinity low salt (150 mM) buffer andinjected into a static loop for loading onto the column. Prior to useboth the BMP2-HBP column and all buffers were equilibrated to roomtemperature for 30 min before washing and equilibrating the column inlow salt buffer for 30 min until all absorbance channels were stable.The sample was then loaded onto the column at 0.5 ml/min and the columnand the sample rinsed in 10 ml of low salt buffer at 0.5 ml/min.Retained GAG+ samples were subsequently recovered by elution with 10 mlof high salt (1 M) buffer and lyophilised for desalting. The column wasthen rinsed in 10 ml of low salt buffer and stored at 4° C.

Pronase/Neuraminidase Treatment

In order to isolate GAG chains from their core proteins, they weredigested using pronase and neuraminidase. Lyophilized PGAG samples wereresuspended in a minimum volume of 25 mM sodium acetate (pH 5.0) andclarified by filtration through a 0.4 μm syringe filter. Total samplevolume was dispensed into 10 ml glass tubes in 500 μl aliquots. 500 μlof 1 mg/ml neuraminidase was added and incubated for 4 h at 37° C. Afterincubation 5 ml of 100 mM Tris-acetate (pH 8.0) was added to eachsample. An additional 1.2 ml of 10 mg/ml pronase, reconstituted in 500mM Tris-acetate, 50 mM calcium acetate (pH 8.0), was added to eachsample and incubated for 24 h at 36° C. After treatment all volumes werecombined and prepared for anion exchange processing by centrifugationand filtration.

GAG Digestion Protocols

The analysis of GAGs, including their sulfated domain sizes and relativesulfation levels, was carried out by using established protocolsincluding degradation by either nitrous acid or lyases.

Nitrous Acid Digestion

Nitrous acid-based depolymerisation of heparan sulfate leads to theeventual degradation of the carbohydrate chain into its individualdisaccharide components when taken to completion. Nitrous acid wasprepared by chilling 250 μl of 0.5 M H₂SO₄ and 0.5 M Ba(NO₂)₂ separatelyon ice for 15 min. After cooling, the Ba(NO₂)₂ was combined with theH₂SO₄ and vortexed before being centrifuged to remove the barium sulfateprecipitate. 125 μl of HNO₂ was added to GAG samples resuspended in 20μl of H₂O, and vortexed before being incubated for 15 min at 25° C. withoccasional mixing. After incubation, 1 M Na₂CO₃ was added to the sampleto bring it to pH 6. Next, 100 μl of 0.25 M NaBH₄ in 0.1 M NaOH wasadded to the sample and the mixture was heated to 50° C. for 20 min. Themixture was then cooled to 25° C. and acidified with glacial acetic acidto pH 3 in the fume hood. The mixture was then neutralised with 10 MNaOH and the volume was then decreased by freeze drying. The finalsamples were run on a Bio-Gel P-2 column to separate di- andtetrasaccharides to verify degradation.

Heparinase III Digestion

Heparinase III is an enzyme that cleaves sugar chains at glucuronidiclinkages. The series of heparinase enzymes (I, II and III) each displayrelatively specific activity by depolymerising certain heparan sulfatesequences at particular sulfation recognition sites. Heparinase Icleaves HS chains within NS regions along the chain. This leads to thedisruption of the sulfated domains that are thought to carry most of thebiological activity of HS. Heparinase III depolymerises HS within the NAdomains, resulting in the separation of the carbohydrate chain intoindividual sulfated domains. Lastly, Heparinase II primarily cleaves inthe NA/NS “shoulder” domains of HS chains, where varying sulfationpatterns are found.

In order to isolate potential active domains we focused on thedepolymerisation of GAG+NA regions. Both the enzyme and lyophilised HSsamples were prepared in a buffer containing 20 mM Tris-HCl, 0.1 mg/mlBSA and 4 mM CaCl₂ at pH 7.5. The concentration of heparinase III addedto each sample is governed by the relative quantity of HS components inthe sample. Our analysis, via nitrous acid depolymerisation, indicatedthat the GAG+ samples consisted of predominantly HS; thus the enzyme wasused at 5 mU per 1 μg of HS. The sample was incubated at 37° C. for 16 hbefore the reaction was stopped by heating to 70° C. for 5 min. Thesample was then applied to the appropriate column system for furtheranalysis.

Cell Culture GAG Production

In order to isolate GAG species representative of developingosteoblasts, MC3T3 cells were grown in osteogenic conditions for 8 days.The cellular component was removed via incubation in a dilute solutionof 0.02 M ammonium hydroxide (NH₄OH) at 25° C. for 5 min. After 5 min,NH₄OH was removed by inversion of the culture surfaces. Treated cultureswere allowed to dry in a laminar flow cabinet overnight. The followingday the treated cultures were washed three times with sterile PBS andallowed to dry in the laminar flow cabinet. Prepared matrix cultureswere then stored under sterile conditions in 4° C. until primaryproteoglycans were liberated via treatment with disruption buffer andanion exchange chromatography.

BMP2-Specific GAG Bioactivity

C2C12 myoblasts were subcultured every 48 h, to a maximum of 15passages, by plating at 1.3×10⁴ cells/cm² in Dulbecco's modified Eagle'smedium (DMEM) supplemented with 10% FCS. Osteogenic differentiation wasinduced at 2×10⁴ cells/cm² in DMEM supplemented with 5% FCS, nominatedconcentrations of recombinant human bone morphogenic protein-2 (rhBMP2)and glycosaminoglycan fractions with a positive or negative affinity forrhBMP2 (GAG+ and GAG− respectively). rhBMP2 and GAG fractions werepre-incubated for 30 min at 25° C. prior to addition to theircorresponding C2C12 cultures. The cultures were permitted to grow underthese conditions for 5 days, with media for each condition being changedevery 48 h, before mRNA samples were extracted and prepared for RQ-PCRanalysis. Real time PCR for osteocalcin expression was conduced usingthe ABI Prism 7000® sequence detection system (Perkin Elmer LifeSciences). Primers and probes were designed using Primer Expresssoftware (v2.1, PE Applied Biosystems). The target probe was redesignedto incorporate LNA bases and labelled with BHQ-1 (Sigma-Proligo). Theribosomal subunit gene 18S (VIC/TAMRA) was used as an endogenouscontrol, with each condition consisting of three repeats, each tested intriplicate. The raw PCR data was analysed using the ABI SequenceDetector software. Target gene expression values were normalised to 18Sexpression prior to the calculation of relative expression units (REUs).

Results Anion Exchange Chromatography

In order to successfully extract GAGs from MX samples, it is necessaryto remove other matrix proteins that may contaminate the sample. As GAGsconstitute the most negatively charged molecules in the ECM, this ismost effectively accomplished with anion exchange chromatography.

Samples were disrupted using 8M Urea/CHAPS buffer and loaded onto theanion exchange column. Unwanted protein and ECM debris were washed fromthe column and the negatively charged GAGs eluted with 1 M NaCl. Atypical chromatogram (FIG. 1) clearly shows the flowthrough of a largeamount of nonadherent debris, as well as the clean and tight elution ofa large quantity of GAGs from the MX preparation. Thus not only doesthis result demonstrate the purification of GAGs by this method, it alsoconfirms the retention of a large number of GAGs in the ECM aftertreatment with NH₄OH.

Desalting

Virtually all chromatography methods employed to purify and analyse GAGsat various stages of processing require elution with high-salt buffers.As high salt conditions interfere with affinity-based chromatography, itis necessary to desalt samples after each stage of processing. Thisprocess is generally completed with size exclusion chromatography. Underthese conditions larger molecules, such as GAGs, exit the column beforesmall molecules, including the salt and small GAG debris. The separationof GAGs from the contaminating salt can be followed on the resultingchromatogram (FIG. 2) which also serves to confirm that the GAG chainsremained intact during the treatment process.

BMP2-HBP Column System Column Preparation

Due to the prohibitive costs involved in creating a BMP2 growth factorcolumn with commercially available reagents, we instead utilised abiotinylated preparation of the known heparin-binding domain of BMP2(BMP2-HBP). This peptide was immobilised on a Hi-Trap Streptavidin HPcolumn (1 ml) in order to specifically retain GAG chains with anaffinity for the specific heparin-binding domain peptide.

First we examined any background affinity the GAGs may have had for thenaked streptavidin column by running the total GAG (tGAG) fractionagainst a column bed devoid of BMP2-HBP (FIG. 3). Our results confirmedthat our MX derived tGAG samples carried no inherent affinity for thestreptavidin column. We further investigated two separate methods ofexposing tGAGs to the BMP2-HBP for the purpose of separating chains witha specific affinity. The peptide was either pre-incubated for 30 minwith 25 mg of tGAGs prior to loading onto the streptavidin column, orwas loaded first, with the tGAGs being run through the column bedthereafter.

Pre-incubation of tGAGs with the BMP2-HBP revealed the completeinability of the peptide to associate with the column (FIG. 4), letalone mediate any isolation of specific GAGs. When the peptide wasloaded onto the column alone, however, its association with the columnwas absolute, with effectively no elution of peptide, even under 1 Msalt conditions (FIG. 5). This high affinity association indicates thatthe biotin-streptavidin association is functioning correctly, andsuggests a possible inhibition of binding to the column, when loadedtogether with tGAGs, due to steric hindrance.

Column Loading Capacities

As the proportion of tGAGs that were likely to have a relative affinityfor the BMP2-HBP was unknown, we first sought to standardise thequantities of tGAGs loaded onto the peptide column at each run forseparation. Hi-Trap columns were prepared by immobilising 1 mg of theBMP2-HBP for the extraction of tGAGs with a specific affinity for theBMP2 heparin-binding site. This amount was selected so as to maximisethe quantity of available peptide for future experiments should columnstability become compromised over time. Instability is a significantproblem with peptide columns, with corresponding impacts on consistency.Initial attempts at loading of 25 mg of tGAGs onto a 1 mg BMP2-HBPcoupled column resulted in a clear overloading, as observed viaabsorbance at 232 nm in the flowthrough (FIG. 6). Although a significantelution peak was observed, tGAGs with affinity for the HBP were lost inthe flowthrough due to overloading. This was examined by re-running theflowthrough through the peptide column (FIG. 7). This resulted in asignificant GAG+ (elution) peak, indicating that the previous run hadsaturated the column.

Further optimisation led us to routinely load no more than 6 mg of tGAGsonto a 2 mg BMP2-HBP column. This, as evidenced by the flowthrough peak(FIG. 8) and the absence of a positive-binding fraction (FIG. 9),forestalled column overloading. The extraction of those tGAGs with anaffinity for the BMP-HBP from each sample set in a single pass allowedus, in turn, to separate GAG+ and GAG− fractions more efficiently.

GAG Domain Analysis GAG+ Chain Specificity

With the establishment of a standardised protocol, we were able toreproducibly isolate GAG+ fractions for further analysis.

Given the domain structure of heparan sulfate that mediates the bindingspecificity for proteins, it is likely that multi-domain GAG chains thatbind to the column are in fact composed of a large proportion of chainwith little or no specific affinity for BMP2. Similarly, it is possiblethat chains that appeared GAG− may in fact contain domains that carrysome affinity for the BMP2-HBP. In order to examine these possibilities,it was necessary to break down the GAG chains into their componentdomains for more extensive examination.

The enzyme heparinase III (heparitinase I) cleaves HS chains primarilyin those areas flanking highly sulfated regions, thereby liberating thehighly charged, protein-associating domains that bind susceptible growthfactors, in this case the BMP2-HBP. Both GAG+ and GAG− fractions wereexposed to heparinase digestion, although neither fraction showed anychange in their affinity for the BMP2-HBP (FIG. 10).

Heparinase III digestion of both full length GAG+ and GAG− fractions wassubsequently conducted, and both digested sample sets subsequentlyloaded onto the BMP2-HBP column to assess retention affinity.

The efficacy of the heparinase digestion was validated by the increasein relative absorbance of samples of equal dry weight after enzymaticdigestion, as shown in FIGS. 10 and 12. As the monitoring of GAG chainsat 232 nm is via the sugar chain itself and, in particular, unsaturatedbonds, any cleavage along the chain's length by heparinase III,resulting in unsaturated bonds of HS fragments, leads to an increase inabsorbance.

Interestingly, heparinase digestion of full length GAG− chains yieldedno fractions carrying any notable affinity for the BMP2-HBP (FIG. 11).However, the digestion of full-length GAG+ samples similarly resulted inno fractions that lacked affinity for the BMP2-HBP (FIG. 12). Thisresult suggests that entire chains of BMP-binding GAG are producedcontaining domain repeats that have a specific affinity for the HBP.Alternatively, the HBP may not be able to yield sufficientdiscrimination between GAG+ domains with varying affinity under theseminimalist conditions.

GAG+ Composition Full Length GAG+ Sizing

In order to examine the composition of GAG+ fractions from the BMP2-HBPcolumn, we first examined their average size. This was to ensure that wewere actually separating GAG chains of reasonable length, rather thansmall fragments not carrying any specific affinity. Although any sizingof GAG chains is problematical, owing to their relatively rigid rod-likeconformation, a set of assumptions invoking Stoke's radius and apparentsphericity can be made.

Full length GAG+ samples were loaded onto Biogel P10 gel filtrationcolumns (1 cm×120 cm) with an exclusion limit of between 20 kDa to 1.5kDa. Absorbance measured at 232 nm indicated a large proportion of GAG+molecules had an overall apparent size greater than 20 kDa (FIG. 13).

It has been posited that sugar chains must be longer than approximately10-14 rings in order to potentiate significant biological activity forthe FGF family of mitogens. In terms of apparent molecular weight, achain of 14 fully sulfated disaccharides corresponds to approximately8.7 kDa. As the majority of chains found in the GAG+ samples show anapparent molecular weight >20 kDa, it is reasonable to assume that theinteraction that they carry for the BMP2-HBP has some specific affinityand is not the result of a general non-specific interaction.

GAG+ Sugar Species

There are five major glycosaminoglycan sugar families: hyaluronan,keratan sulfate, dermatan sulfate, chondroitin sulfate and heparansulfate. Of these five, only heparan sulfate, chondroitin sulfate anddermatan sulfate have the capacity to generate variably sulfated domainsthat may code for specific interactions with particular cytokines suchas BMP2. The identification of the type of sugar species isolated usingthe BMP2-HBP column was of crucial importance for this study, and wasdetermined using a combination of diagnostic chemical and enzymaticdegradations. In particular, heparan sulfate, one of the major GAGcandidates for the interaction with BMP2, can be completely degradedinto its disaccharide components in the presence of nitrous acid.

Thus, our HBP-retained GAG samples were incubated with nitrous acid for20 min prior to separation on a Biogel P10 sizing column. Examination ofthe resulting chromatogram revealed an almost complete degradation ofall GAG+ sugar samples, as measured by absorbance at 232 nm and 226 nm(FIG. 14).

This result strongly suggests that the full length sugar chains isolatedspecifically against the BMP2-HBP consist primarily of heparan sulfate,as other sugar chains are not affected by nitrous acid depolymerisation.

Although almost all the GAG+ chains could be degraded in such a manner,a small peak was nevertheless observed at higher molecular weights (>20kDa). It can be postulated to consist of chondroitin sulfates, of whichCS-B (dermatan sulfate) and CS-E (chondroitin-4,6-sulfate) demonstratesulfation complexity akin to heparan sulfates.

GAG Species Analysis BMP2-HBP Specific GAGs (Alternative Species)

The degradation of full length GAG+ chains by exposure to nitrous acidclearly indicated that the majority of GAG+ sugar chains consisted ofthe heparan sulfate sugar species (FIG. 14). The degradation of the GAG+sample was not, however, complete as was observed by the remnant peak inthe high molecular weight region. The presence of this peak pointsstrongly to the possibility of other species of sugar chains, such aschrondroitin or dermatan sulfate. We next sought to examine the possibleaffinity the other two sugar types may have for this cytokine by firstexamining a variety of commercially available chondroitin and dermatansugars for their affinity to the BMP2-HBP column.

We tested chondroitin-4-sulfate (C4S), chondroitin-6-sulfate (C6S) anddermatan sulfate (DS) by, in each instance, loading 6 mg of the sugaronto the BMP2-HBP column under the same conditions used to isolate GAG+chains from MC3T3 matrix samples.

The chromatograms illustrating the affinity of each of the 3 sugar chaintypes showed that only C4S (FIG. 15) had any significant affinity forthe peptide. This affinity taken together with the lack of affinity forthe BMP2-HBP column observed for both C6S (FIG. 16) and DS (FIG. 17)samples, appears to indicate that C4S has a particular, potentiallysignificant, interaction with the BMP2 heparin-binding site.

As any potential interaction between chondroitin sulfate and BMP2 hasnot yet been well characterised, these results led us to question thevalidity of column chromatography as an accurate monitor of theBMP2/heparan interaction. In order to further explore the specificity ofthe interaction dynamic, we tested several commercially available sugarspecies for their affinity to the column. These included heparansulfate, low molecular weight heparin (Heparin-LMW), high molecularweight heparin (Heparin-HMW) and Heparin-HMW treated with heparinase I.

Interestingly, none of these commercially available GAG species appearedto demonstrate any specific interaction with the peptide column. Heparansulfate from bovine kidney had very little affinity (FIG. 18), abehaviour that was further confirmed by its inability to positivelyaugment FGF2-mediated cell proliferation (data not shown), as isobserved in the presence of HS2. This reduced ability of this GAG sampleto bind the column may be as a result of it being sold in a relativelyunsulfated form.

None of the tested heparin samples showed even a minor affinity for thecolumn. This is of particular interest as BMP2 itself was historicallyfirst isolated using heparin columns. In order to confirm this result,both LMW (FIG. 19) and HMW (FIG. 20) heparin were tested; neither showedany appreciable affinity for the column.

As we surmised that the relatively small BMP2-HBP peptide may have haddifficulty maintaining its association with the much larger heparinmolecules, we next predigested the heparin-HMW samples using heparinaseI. These smaller heparin-HMW fragments were then run over the BMP2-HBPcolumn; this treatment did not, however, appear to improve the abilityof any of the heparin samples to bind the peptide column (FIG. 21).

This inability of the peptide column to show any specific interactionwith any of the various preparations of heparin was somewhat unexpected,due to BMP2 conventionally being isolated via heparin affinity. It ispossible, however, that this may be as a result of the reversing of the“receptor-ligand” order of interaction; in this case the BMP2-HBPrepresented the fixed “receptor” as opposed to the heparin thatrepresented the “ligand”, or that the concentrations of BMP2-HBP orsoluble heparin favour a dissociated state that rapidly negates anyaffinity under flow/salt stress.

Conclusions

The use of a preosteoblast-derived ECM substrate provided us with auseful model for simulating the activity of natively secreted,ECM-associated GAGs in relation to such osteoinduction. Though numerousprevious studies have examined the role that this native interaction hasin modulating the activity of BMP2, this has usually been conducted atthe level of the cytokine, rather than with a view to exploring thesequence specificity of the biomodulating GAGs.

Hence here we sought to exploit the availability of natively secretedGAGs in the MX substrate and their potential for direct,sequence-specific interaction and modulation of BMP2-induced C2C12myoblast commitment to the osteogenic lineage.

Anion Exchange

The use of this particular standard and well characterised protocolprovided us with conclusive evidence for GAG accessibility from theNH₄OH-treated MX substrate. Our initial concerns were centred around theharsh chemical treatment used to lyse the cellular components of theECM, and that this may have also resulted in the stripping of themajority of GAGs from the ECM. However, the significant, high affinitypeak observed in the anion exchange chromatogram clearly illustrates theretention of a large quantity of GAGs within the MX substrate. Whilethis particular methodology does not allow for the identification ofindividual GAG species, it does offer conclusive evidence of theirpresence in the sample due to their being amongst the mostnegatively-charged molecules secreted by cells.

BMP2-HBP Column System

Previous research into the functional role of the BMP2 heparin-bindingpeptide provided us with a useful tool to investigate the potentiallyspecific interaction that BMP2 has with GAGs. This single string ofamino acids, located at the N-terminus of each BMP2 monomer, appears tobe solely responsible for mediating BMP2's affinity for GAGs.

We thus investigated the use of this region of the BMP2 molecule as aligand “bait” in attempts to retain those GAG chains that carriedrelative affinity for the cytokine. The use of the BMP2-HBP in thismanner resulted in a significant retention of HS to the peptide column(GAG+).

Column Preparation

Using an N-terminal biotinylated HBP we prepared a BMP2-HBP affinitychromatography column, and were able to successfully retain GAG samplesthat were candidates for controlling the native BMP2 homodimer. Initialpreparations of the column highlighted some interesting problems.Preparations of biotinylated BMP2-HBP that were premixed with tGAGsshowed an inability to bind to the column. As later tests showed thatthe BMP2-HBP easily attached to the streptavidin column when loaded onits own this result indicated that the GAGs interfered with the abilityof the peptide's biotinylation site to associate with the streptavidincolumn. The tGAGs themselves carried no affinity for the streptavidin,indicating that the direct interaction with the BMP2-HBP, possibly viasteric hindrance, was responsible for this.

Column Optimisation

Without any direct information that would allow us to estimate thebinding capacities of GAG+ sugars in our samples, our peptide columnneeded to be optimised to ensure that excessive sample loading would notlead to column saturation and consequent sample loss. This initiallyinvolved intentionally saturating the column in order to examine thebinding capacity of a known quantity of BMP2-HBP. Even with a largequantity of tGAGs the peptide was capable of retaining the majority ofGAG+ sugar chains. Under these conditions as little as 1 mg of BMP2-HBPwas able to completely retain all GAG+ chains within two cycles. Thecolumn thus appeared to “simulate” a true BMP2 growth factor column andprovide an extremely efficient way of extracting GAG+ samples.

The optimisation of peptide-based columns for specific GAG isolation isa complex procedure that varies greatly depending on the size andindividual chemical characteristics of the protein used. Previousstudies, utilising FGF-1 and 2 growth factor columns (Turnbull andNurcombe, personal communication), also showed a significant need forcontinual column maintenance and short viable column life-spans. Thesestudies demonstrate the laborious nature of working with peptide columnsand the care that must be taken to correctly optimise this manner ofsystem. Unfortunately, while other systems for the analysis of specificprotein-GAG interactions exist, these generally lack the capacity toisolate sufficient quantities of GAGs for further analysis, making theminappropriate for our intended course of study.

GAG Domain Analysis

GAG sulfation patterns are, particularly in the case of heparan sulfate(HS), frequently concentrated into domains of high sulfation that areinterspaced with regions of little sulfation. This grouping of sulfationsites into domains is what provides region-specific binding of ligandsto the GAG chain, allowing a single sugar molecule to potentially bind avariety of different targets, and to stabilise the interaction betweenthese, as is seen in the FGF system. Exceptions to this proposed modelfor HS-ligand interactions include the interaction between interferongamma (IFNγ) and heparan sulfate. In this instance the interactionbetween the GAG and IFNγ leads to an increased potency of the cytokine.IFNγ that remains dissociated from local GAGs is rapidly processed intoan inactive form, thereby preventing its signalling in inappropriateareas after diffusion. IFNγ also displays four separate heparin-bindingdomains, each with a different sequence, a finding not unusual forheparin-binding proteins. However, only two domains found immediately atthe C-terminus of the protein have been shown to mediate INFγ'sheparin-binding characteristics. Importantly, sequence analysis of theHS sequence with specific affinity for these two IFNγ heparin-bindingsites revealed an interesting difference in comparison to the commonlyobserved model of HS-ligand interaction. In this case, the sequence ofHS responsible for the binding of IFNγ was found to be composed of apredominantly N-acetylated region, carrying little sulfation. Thisregion was flanked by two small N-sulfated regions. This differssignificantly with the system observed in FGF, where sulfation patternsin NS domains are responsible for mediating the interaction between FGFand HS. In recent years, this type of interaction has been observed innumerous other systems, such as PDGF, IL-8 and endostatin. The discoveryof this kind of interaction with HS, as observed in these cytokines, maybe able to explain the bioactivity observed in hyaluronan, which carriesno sulfation patterns at any point along its chain and yet has theability to modulate the activity of such factors as NF-κB.

These observed interactions between ligands and GAGs, in particular thatof IFNγ, differ significantly to the proposed, and our observed, mode ofinteraction between HS and BMP2. BMP2's single, N-terminalheparin-binding domain exhibits no secondary structure and appears tointeract with HS solely on the basis of charge. While in-depth sequenceanalysis of HS that binds this peptide sequence was not conducted, itsrequirement to be eluted under approximately 300 mM NaCl conditions leadus to suspect the presence of a moderate degree of sulfation, therebyplacing this interaction within the conventional model of sulfationpatterns mediating specific interactions.

GAG+ Chain Specificity

The allocation of sulfation patterns into domains that give HS itsability to stabilise proteomic interactions also results in thepossibility that a GAG+ sugar chain of sufficient length and complexitymay carry several domains that have no direct affinity for the BMP2-HBPon their own, due to their carrying a different sulfation sequence.Conversely, it is also possible that some full-length sugar chains thatwere identified as having little affinity for the BMP2-HBP (GAG−) maycontain some cryptic domains that do carry such affinity.

In recent years, numerous reports have been published that providestrong evidence for a “sulfation code” within these complex carbohydratechains. While the details of this “sulfation code” remain difficult toelucidate, and the sequencing of long chains of sulfated carbohydratesis a complex and time consuming process, a number of possible modes ofspecific interaction between GAGs and ligands have been proposed. Oneobservation in particular has led to the characterisation of numerousGAG-ligand models; the grouping of sulfation into discrete regions, or“domains”, along the length of many types of GAGs, such as heparansulfate. Interestingly no template for this phenomenon has yet beenobserved, and it appears to be primarily a result of the temporalactivity of the sulfotransferase enzymes responsible for this phase ofGAG synthesis.

Particularly useful tools in the study of specific GAG sequences are anumber of heparin lyases that can be used to examine targeteddepolymerisation of complex carbohydrate chains, thereby providinginsight into their structure. One particular heparan lyase, heparinaseIII (heparitinase), cleaves heparin sulfate chains at sites flanking thehighly sulfated domains that may occur in heparan sulfate chains. Thus,using this enzyme, it is possible to liberate these potentially activeregions from the full length sugar chains and separate them, if theyfunction as single domains, via affinity chromatography, from regionswith no specific affinity for the BMP2-HBP.

It is important to note that, in the case of GAG-ligand interactions,affinity by sequence does not necessarily guarantee bioactivity. Themode of activity mediated by GAGs during their association with theirvarious ligands differs greatly depending on the system. In someinstances where the sugar chain is responsible for prolongingprotein-protein interaction via stabilisation of tertiary proteinstructures, such as is found between FGF and its receptor, and theinteraction between HGF/SF and Met, multiple discrete sulfation regionsmay be involved in mediating the intended bioactivity of the sugarchain. In such instances the isolation of individual sulfated domainsfrom a full length carbohydrate chain may, in fact, result in aninhibition of sugar bioactivity since though each “domain-fragment”still binds its intended target it is unable to mediate the intendedbiological effect of a combined full length carbohydrate chain.

Interestingly, this particular characteristic of GAG-ligand interactionsis precisely what makes this manner of approach useful for modulatingBMP2 activity. The proposed model for GAG modulation of BMP2 bioactivityinvolves immobilization of the cytokine to GAGs in the ECM or on thecell surfaces. In this type of system the application of exogenous GAGsspecific to the heparin-binding domain of BMP2 would prevent thisinteraction, increasing short term BMP2 mediated signalling, similar tothe effect observed during the addition of soluble heparin. While thereis some indication that this manner of interaction would continue toprotect the cytokine from proteolytic degradation, delocalization ofBMP2 from its intended region of bioactivity has the potential tonegatively impact the cytokines effectiveness in the long term.

Control testing of our full length GAG+ and GAG− chains resulted insimilar profiles to those observed during their primary separation.Analysis of GAG+ and GAG− chains post treatment with heparinase III,however, gave surprising results. The digestion of GAG+ chains did notseem to generate separable fragments based on simple affinity for theBMP2-HBP. Furthermore, the digestion of full length GAG− chains yieldedno liberation of positive domains from the negative sugar chains. Thereis some possibility that the enzymatic digestion did not go tocompletion. However, the resulting chromatogram clearly showed a largeincrease in the absorbance at 232 nm when compared to the full lengthGAG chains. As a large proportion of the absorbance ofglycosaminoglycans at 232 nm is mediated via absorbance of unsaturatedbonds, such as those formed during enzymatic depolymerisation, itstrongly indicates that the enzymatic digestion was, in fact,successful.

The implications of this result are somewhat unusual. This data suggeststhat GAG chains are not only synthesised by cells to specificallyinteract with BMP2, but that, in the case of MC3T3 cells, these sugarchains carry a number of sequence repeats specific for aspects of BMP2metabolism. The fact that BMP2 is an extremely potent factor may offeran explanation for this observation. The effects of BMP2 on theosteoinduction of mesenchymal progenitor cells is well documented, as isits ability to induce ectopic bone formation in cells that are even moreremoved from the osteogenic lineage. Given this potency, aberrantsignalling of BMP2 is known to have deleterious consequences both forhealing and in development. It is possible that numerous repeats of theBMP2-HBP interaction sequence on preosteoblast GAGs are designed toensure a maximal binding, and thereby the modulation, of this cytokine'sability to induce altered cell fate. Conversely, the extremely lowconcentrations of BMP2 produced in vivo may also require this type ofsugar chain production in order to ensure the retention of a sufficientlocal concentration, an observation supported by the extremely highconcentrations of BMP2 required in vitro to induce the osteogenicdifferentiation of C2C12 myoblast cells.

Of particular interest is the fact that this repetition of BMP2-bindingdomains is produced via a synthesis pathway for which no template ortiming mechanism has yet been elucidated. The accuracy andreproducibility of sequence specific domains within a single sugar chain(as opposed to the random clustering of such domains with those againstother ligands) strongly suggests that these cells do, in fact, have theability to direct the generation of specific sugar sequences. Thecurrent understanding of HS structure implicates the progressivepost-synthesis “editing” of the carbohydrate chain in the generation ofsequence-specific regions, with observations pointing towards somemanner of enzymatic “template”, whereby the local concentrations ofparticular sulfotransferases as well as other interacting molecules areused to directly control the generation of specific sugar sequences. Ourcurrent understanding of this mode of specific synthesis is largelyformulated based on numerous studies including those by Lindahl et al.that investigated the high affinity interaction between antithrombin IIIand heparin, and those by Esko et al. involving Chinese hamster ovary(CHO) cell mutants with altered GAG synthesis pathways. These studies,while varying significantly in their approaches to GAG analysis, allpoint towards a highly conserved system of specific GAG synthesis, forthe directed modulation of cytokine and receptor activity. Importantly,these studies also serve to explain the potential generation of suchBMP2 repeats as were observed in our study.

GAG+ Constitution Full Length GAG+ Size

The bioactivity of individual GAGs chains for FGFs is closely related tocarbohydrate chain length. A common approach to assessing GAGbioactivity is to assay ever shorter sulfated domain fragments and sodetermine the shortest possible sequence required to mediate theactivity observed.

Using this approach we first examined full length GAG+ sugar chains, anddetermined that they were >20 KDa in size, long enough to carry multipledomains with affinity for BMP2. Interestingly, this observation providedsupport for the earlier observation that GAG+ samples treated withheparinase 3 showed multiple repeats of carbohydrate chain segments witha specific affinity for BMP2, since a variably sulfated sugar chain ofthis size has the capacity to carry numerous sulfated domains.

GAG+ Sugar Species

With the majority of the five glycosaminoglycan types that constitutethe “glycome” able to encode the observed specific interactions withBMP2, it was necessary to elucidate which of these GAG types could beinvolved in this specific association. Although the prime candidate forthis interaction is a heparan sulfate, analogous growth factorinteractions have also been identified for chondroitin and dermatansulfates.

Heparan sulfate can be totally depolymerised into its disaccharidecomponents with nitrous acid. This particular characteristic, sharedwith heparin and keratan sulfate, is essential for the analysis ofspecific GAG populations. In the case of our analysis of thecarbohydrate constituents of our GAG+ samples, degradation due tonitrous acid was diagnostic of heparan sulfate. This probability isprimarily due to its heparan sulfate's higher degree of chargepatterning via sulfation in comparison to either heparin or keratansulfate. Ultimately, this charge patterning is responsible for BMP2'sspecific interaction with HS.

Our analysis utilising the nitrous acid protocol showed a completedegradation of the GAG+ sample set indicating that the majority ofsugars in the GAG+ sample set were in fact 1,3-linked and, thus, wereheparan sulfate. This result supports the numerous observations inregards to the specificity of heparan sulfate cytokine interactions,particularly the interaction that BMP2 exhibits with heparin and HS.

GAG Species Analysis BMP2-HBP Specific GAGs (Alternative Species)

The small remnant peak that was observed after the degradation of GAG+samples by nitrous acid supports the possibility that other sulfatedGAGs carrying some specific affinity for BMP2 may be found in the GAG+sample set. Given our current understanding of the role of sulfation inmediating the interaction between GAGs and BMP2, chondroitins anddermatans are the most likely alternative sugars to show a specificinteraction with BMP2 as these show the highest potential diversity insulfation patterns.

A methodology frequently employed for GAG analysis includes examiningthe role of individual sulfation positions on GAG-ligand interactions.This method of analysis gives an indication of the importance ofindividual sulfation positions in maintaining the interaction betweenthe GAG chain and its specific target. Furthermore, since the differentspecies of GAGs only have the potential to carry sulfation patternsspecific to their species, this can aid in narrowing the possibleglycosaminoglycan candidates that may show an affinity for a specificligand.

To this end we examined the affinity for the BMP2-HBP carried byvariably sulfated CS chains, C4S and C6S, and standard DS.Interestingly, only C4S carried any significant affinity for theBMP2-HBP. This data indicates that it is likely that the 4-O-sulfationis necessary for CS to interact with the BMP2-HBP. Interestingly,dermatan sulfate showed no affinity for the BMP2-HBP. This observationis of interest since DS is the only CS species that demonstratesdiversity in sulfation similar to that of HS. Furthermore, ourobservations indicate a possibility that the epimerisation of GlcA toIdoA in DS compromises the ability of this sugar type to bind theBMP2-HBP. Both C4S and DS are able to carry 4-O-sulfation, yet onlysmall quantities of DS were retained on the column in comparison to C4S.Alternatively, this lack of affinity may simply be due to thisparticular batch of DS not carrying sufficient 4-O-sulfation toeffectively mediate binding to the BMP2-HBP. Interestingly, theseparticular observations appear to demonstrate an interaction betweenBMP2 and CS carrying 4-O-sulfation. While previous studies haveinvestigated the use of CS-BMP2 interactions in drug delivery systems,not much is known about any sequence specific interaction betweenindividual CS species and BMP2. However, since HS chains are composed of1,4-linked disaccharide units, the observed 4-O-sulfation responsiblefor CS-BMP2 interactions is not found in HS-BMP2 interactions, pointingto a sequence specific interaction not found in CS. Thus it is likelythat the remnant peak observed post-nitrous acid treatment may containsmall quantities of 4-O-sulfate carrying C4S or DS.

Further investigation revealed that neither commercial HS nor heparinheld any significant affinity for the peptide column. The HS used forthis assay was purchased commercially from Sigma-Aldrich and was derivedfrom bovine kidney. Given what is known about the tissue specificity ofHS it is possible that this commercially available HS, isolated frombovine kidney sources, carried negligible carbohydrate sequencesrequired to specifically mediate an interaction with BMP2. Similarlyneither LMW nor HMW heparin showed any affinity for the peptide column.The heparin used for this analysis was also purchased fromSigma-Aldrich, and was derived from porcine intestinal mucosa.

While heparin's interaction with antithrombin III has been wellcharacterised, and notwithstanding its versatile role in the isolationof susceptible molecules, heparin's interaction with growth factors isnot, in general, regarded to be specific due to its uniform sulfation.However, given that heparin is routinely used to isolate BMP2, it issomewhat surprising that neither of the heparin samples interacted withthe peptide column to any significant degree.

A further possibility for this lack of interaction between the peptidecolumn and heparin is due to the difference in molecular weights betweenthe two molecules. The small BMP2-HBP attached to the column may havedifficulty in maintaining its association with the larger, heavilysulfated heparin chain. The inability of heparinase-cleaved heparin tobind the column, however, appeared to indicate that the steric effectsof using full length heparin on the column were not solely responsiblefor disrupting the potential interaction between the sugars and theBMP2-HBP. There is no immediately apparent reason for this inability forcommercial heparin to associate with the BMP2-HBP column, though it maybe postulated that further spatial separation of the BMP2-HBP from itsassociated bead via spacer chains may help to ameliorate this problem.

SUMMARY

In this study we have demonstrated the use of affinity chromatography toisolate a subset of glycosaminoglycans that carry a specific affinityfor the BMP2-HBP, and have shown the potential for this procedure toyield reproducible results. During this portion of our investigationinto the interaction between matrix based GAGs and BMP2, we have madeseveral observations with regards to both the type of GAGs involved inmediating this association and their structure.

Our results have implicated heparan sulfate for mediating the majorityof the affinity BMP2 has for the preosteoblast ECM, an interaction whichis increasingly recognised as being responsible for the modulation ofBMP2 activity. Furthermore, our investigation into the likely structureof the ECM-resident GAGs isolated on the basis of their affinity for theBMP2 heparin-binding site have yielded a surprising result.

Our data indicates that full length BMP2 GAG+ chains do not consist ofindividual domains with specific affinity for BMP2 interspersed withregions of little or no affinity for the factor. Instead, our resultsimply that these GAG+ chains consist of multiple BMP2-binding domainrepeats. This result is surprising on several levels. Firstly, therepetition required to fulfil this observation over the full length ofa >20 kDa carbohydrate chain points to the presence of some manner ofsynthetic template. Indeed, while previous studies have been unable toderive a template for the assembly of tissue-specific GAG chains, thevery fact that such specificity exists supports the presence of atemplate-based system. Although no genomic template has been elucidatedfor this process there exists some possibility of a proteomic, perhapsenzymatic, template.

Secondly, this observation provides some evidence as to the importanceof the interaction between BMP2 and GAGs. Multiple repeats of the BMP2affinity site along the length of the carbohydrate chain may be requiredto ensure maximal binding of BMP2 to the ECM. This particularassociation has been shown to significantly lengthen the factor's halflife, as well as probably being responsible for maintaining asignificant local concentration in order to maintain signalling.Alternatively, some studies have proposed a model whereby BMP2 isspatially inhibited from interacting with its receptors due to theinteractions with ECM-based GAGs. In this particular scenario therepetition of BMP2 affinity sequences would ensure a maximal binding ofthe factor, thus reducing the chance of it interacting with itsreceptors.

Our cumulative results indicated that this system for the isolation ofGAGs from the ECM is viable and likely to yield GAG chains that have aspecific affinity for BMP2.

This study supports previous findings in regards to the interactionbetween GAGs and BMP2. Although the prevention of BMP2 associating withthe ECM in vitro through the addition of exogenous GAG+ appears toincrease BMP2 signalling and upregulates osteogenic gene expression,observations to the contrary have also reported. In these studies, invivo examination of BMP2's modulation via the HBP showed a distinctimprovement in long term osteogenesis when the association with ECM GAGswas increased. It is possible that this interaction plays a major rolein maintaining local concentrations by preventing the factor fromdiffusing away from its sites of primary activity. In light of thesestudies and our own observations, we propose that BMP2's activity isboth positively and negatively regulated by its association with GAGs.Negative regulation may occur precisely via the model proposed byKatagiri and colleagues, whereby the retention of BMP2 in the ECM, awayfrom its receptors, leads to a downregulation of BMP2 signalling.However, cells that require signalling by this factor may potentiallysecrete various enzymes to remodel extracellular sugar chains, such assulfatases and heparinases, in order to “clip away” GAGs retaining BMP2in the ECM, thereby liberating the factor and allowing it to signal,leading to the BMP2-ECM interaction ultimately becoming one of positivemaintenance of the cytokine's activity. Alternatively, negativeregulation of BMP2 by cell surface GAGs, may be via the internalisationof GAG chains with their associated BMP2 molecules, as has been observedby Jiao and colleagues.

These previous studies, in conjunction with our own observations, havelead us to conclude that the sequence-specific interplay between BMP2and heparin sulfate represents an intricate control mechanism that hasthe capacity to both positively and negatively regulate BMP2 signaling.Physiologically this interaction is responsible for enforcing contextdependent responses to this potent cytokine in respect to many facets ofembryonic development, precursor commitment and wound healing.

Example 2 Purification of BMP2 Peptide Specific HS

We used a peptide having heparin-binding properties from the matureBMP-2 sequence to identify novel HS that bind to the peptide.

Mature BMP-2 Amino Acid Sequence:

[SEQ ID NO: 5] QAKHKQRKRLKSSCKRHPLYVDFSDVGWNDWIVAPPGYHAFYCHGECPFPLADHLNSTNHAIVQTLVNSVNSKIPKACCVPTELSAISMLYLDENEKVVL KNYQDMVVEGCGCR

Heparin-Binding Peptide Amino Acid Sequence:

QAKHKQRKRLKSSCKRHP [SEQ ID NO: 1]

To replicate the natural presentation of the heparin-binding site webiotinylated the peptide on it's C-terminus and kept the proline (P) toimprove the flexibility/accessibility of the peptide once bound to thestreptavidin column.

Isolation of BMP2 Peptide Specific HS

Materials used included a BMP2—peptide coupled Streptavidin column,HiPrep Desalting Column (GE Healthcare), 20 mM PBS+150 mM NaCl (Low SaltBuffer), 20 mM PBS+1.5 M NaCl (High Salt Buffer), HPLC grade Water(Sigma), Biologic-Duoflow Chromatography system (Bio-Rad) and a FreezeDrier.

The column was equilibrated with Low Salt buffer and 1 mg SigmaHS(H9902) was dissolved in low salt buffer and passed through theBMP2-Streptavidin column. Unbound media components were removed from thecolumn by washing low salt buffer (20 mM PBS, pH 7.2, 150 mM NaCl) untilthe absorbance of the effluent at 232 nm almost return to zero. HS boundto the matrix was eluted with high salt buffer (20 mM PBS, pH 7.2, 1.5 MNaCl). Peak fractions were pooled and freeze dried for 48 hrs.

HS 1 mg was applied to the column and washed with 20 mM PBS buffercontaining a low (150 mM) NaCl concentration. After washing with lowsalt buffer, the bound HS were eluted with 20 mM PBS buffer containing ahigh (1.5 M) NaCl concentration. Peaks representing retained fractions(monitored at 232 nm) were collected and subjected to further desalting.

After freeze drying 6 mg of positive HS (GAG+) and 1.8 mg of negative HS(GAG−) were obtained.

Example 3 Evaluation of BMP-2 Specific Heparan Sulfates

C2C12 are mouse mesenchymal stem cells normally exhibiting myogenicdifferentiation but capable of being directed in the osteogenic lineagewith supplementation of BMP-2 at passage 3. C2C12 cells at passage 3were maintained in DMEM with 1000 g/L glucose (low glucose), 10% of FCS,1% of P/S and without L-glutamine (maintenance media).

DMEM with 1000 g/L glucose (low glucose), 5% of FCS, 1% of P/S andwithout L-glutamine was used as differentiation media.

Effect of BMP-2 on Osteogenesis

We evaluated the effects of exogenous BMP-2 on osteogenesis by measuringthe levels of expression of osteogenic markers (osteocalcin, osterix,Runx2).

Through assaying the effect of addition of different amounts (100 ng/mland 300 ng/ml) of BMP-2 to the cells we observed a significant decreaseat day 5 in the expression of osterix, osteocalcin and Runx2 in cellshaving 100 ng/ml BMP-2 compared to addition of 300 ng/ml BMP-2 (FIGS.27-29). Thus we chose this time point for future tests, as any changesshould be readily observable.

Materials and Methods

C2C12 cells at passage 3 were used. Cells were kept in liquid Nitrogenat Passage 3 with 1×10⁶ cells/vial. Once cells were taken from liquidNitrogen, we added 500 μl of culture media, pipetted up and down torefreeze the cells and immediately added 15 ml of culture media.

Culture media was DMEM with 1000 g/L glucose (low glucose), 10% of FCS,1% of P/S and without L-glutamine. Treatment media was DMEM with 1000g/L glucose (low glucose), 5% of FCS, 1% of P/S and without L-glutamine.

C2C12 cells were allowed to grow to 75% confluence before harvesting(normally 2 to 3 days) in culture media.

Cells were counted as follows. Media was first aspirated/discarded; 15ml of PBS added, discard the PBS and add 3 ml of trypsin, incubate at37° C. for 5 min to lift the cells from the flask. 9 ml of culture mediaadded to neutralize the trypsin. GUAVA used to determine the amount ofcells for subsequent cell seeding onto the experiment plates. Forexample, for 3 sets of 12-well plates 30,000 cells×36 wells×3.7cm²=4,000,000 cells. Dilute the cells from the stock and add the desiredamount of culture media for cell seeding (each well requiring 500 μl ofmedia with 30,000 cells).

To prepare BMP2 stock 10 μg rhBMP2 (Bone Morphogenetic Protein 2) wasre-suspended in 100 μl of 4 mM HCl/0.1% BSA.

The following RNA extraction protocol was used. 350 μl of RA1 buffer wasused for cell lysis. Cells were frozen with RA1 at −80° C. for one dayafter which cells were thawed and the lysate filtered for 1 min at11,000 g. The filtrate was mixed with 350 μl 70% ethanol in 1.5 ml tubesand centrifuged for 30s at 11,000 g. 350 μl of MDB buffer was added andthe mixture centrifuged for 1 min at 11,000 g. 95 μl of Dnase reactionmixture added and mixture left at room temperature for at least 15 min.Then wash with 200 μl of RA2 buffer (to deactivate the Dnase), andcentrifuge for 30s at 11,000 g. Wash with 600 μl of RA3 buffer,centrifuge for 30 second at 11,000 g.

Wash with 250 μl of RA3 buffer, centrifuge for 2 min at 11,000 g. Elutethe RNA with 60 μl of Rnase-free H₂O, centrifuge for 1 min at 11,000 g.Measure the concentration using Nanodrop (unit in ng/μl).

RT (reverse-transcription) experiments were performed as follows. Thefollowing were mixed in a PCR tube: Random Primer (0.1 μl), DNTP (1 μl),RNA (250/500 ng), Rnase-Free H₂O (topped up to a final volume of 13 μl).Incubate at 65° C. for 5 min. Incubate on ice for at least 1 min.Collect the contents and centrifuge briefly before adding: 1^(st) StrandBuffer (4 μl), DTT (1 μl), RnaseOUT (1 μl), SSIII Reverse (1 μl). Top upto final volume of 20 μl. Mix by pipetting up and down. Incubate at roomtemperature for 5 min. Incubate at 50° C. for 60 mins. Inactivate thereaction at 70° C. for 15 min.

Reverse-transcription experiments were performed twice on separate daysand the PCR products pooled together and diluted to a finalconcentration of 2.5 ng/μl for subsequent Real-Time PCR.

The Real-Time PCR was performed using a TaqMan® Fast Universal PCRmaster Mix (2×) (Applied Biosystem). PCR master Mix (10 μl), ABI probe(1 μl), cDNA (1 μl), ddH₂O (8 μl). GAPDH and Beta actin were used ascontrol genes against the experimental targets OSX (osterix), OCN(Osteocalcin) and Runx2.

Effect of BMP-2 Specific HS GAG+ on Osteogenesis

We evaluated the effects of the BMP-2 specific HS (GAG+) isolated inExample 2 on osteogenesis by measuring the levels of expression ofosteogenic markers (osterix, Runx2, alkaline phosphatase and BspII) byquantitative polymerase chain reaction (qPCR). A time course wasprepared to compare the expression of the markers over a course of 10days to compare the control to a low and a high dose of BMP-2, the highdose being the optimal conditions to induce differentiation of thecells.

Materials and Methods

Cells were seeded at 30,000 cell/cm² in maintenance media and left toattach overnight. The following day we switched to differentiation mediawith:

-   -   No additives    -   100 ng/mIBMP-2 (positive control)    -   100 ng/mIBMP-2+30 μg/ml−GAG (Neg GAGs)    -   100 ng/mIBMP-2+30 μg/ml+GAG (Pos GAGs)    -   100 ng/ml BMP-2+30 μg/mlHeparin (Sigma # H3149)    -   100 ng/mIBMP-2+30 μg/mlTotal Heparan Sulfate (Sigma # H9902—HS        prior to fractionation)

The carbohydrates and BMP-2 were mixed together in the smallest volumepossible and incubated at room temperature for 30 minutes before theiraddition to the media and on the cells.

After 5 days, RNA was extracted using the Macherey-Nagel kits andReverse-Transcription was performed.

As we show in FIGS. 30-33, the Heparan sulfate from porcine mucosa(Total HS) can increase the activity of BMP-2 (shown through GAG+induced increases in the expression of Alkaline Phosphatase, osterix,BspII and Runx2) and this activity is contained within the fraction thatbinds BMP2 (Pos GAGs). This means that we can isolate the BMP enhancingfraction of a commercial HS by passing them on the BMP-HBD peptidecolumn.

Example 4

MC3T3-E1 (s14) preosteoblast cells (a mouse embryo calvaria fibroblastcell line established from the calvaria of an embryo) were expanded inaMEM media supplemented with 10% FCS, 2 mM L-glutamine, 1 mM sodiumpyruvate and Penicillin/Streptomycin every 72 hours until sufficientcells were generated for plating. The cells were differentiated byplating at 5×10⁴ cells/cm² in aMEM media supplemented with 10% FCS, 2 mML-glutamine, 25 μg/ml ascorbic acid, 10 mM β-glycerol phosphate andPenicillin/Streptomycin. The media was changed every 72 hours for 8 daysat which point the cells and media were harvested. The media wasretained and clarified by high speed centrifugation and filtrationthrough a 0.4 μm filter. The cell layer was disrupted using a cellscraper and an extraction buffer containing PBS (150 mM NaCl w/o Ca²⁺and Mg²⁺), 1% CHAPS, 8 M Urea and 0.02% NaN₃.

At all stages (unless otherwise stated), samples were clarified beforeloading onto column systems. This process included high speedcentrifugation at 5000 g for 30 min, and filtration through a 0.4 μmsyringe filter. The samples were always clarified directly prior toloading through the column system to prevent precipitates forming instagnant solutions.

Anion exchange chromatography was used to isolateproteoglycosaminoglycan (PGAG) fractions from both the media and celllayer samples. In each case, the media or cell layer samples were runthrough a Pharmacia XK 26 (56-1053-34) column packed with Capto Q AnionExchange Beads (Biorad) at a flow rate of 5 ml/min on a Biologic DuoFlowsystem (Biorad) using a QuadTec UV-Vis detector. The samples were loadedin a low salt buffer containing PBS (150 mM NaCl w/o Ca²⁺+ and Mg²⁺),100 mM NaCl, 0.02% NaN₃ at pH 7.3. The samples were eluted in a highsalt buffer containing PBS (150 mM NaCl w/o Ca²⁺ and Mg²⁺), 850 mM NaCland 0.02% NaN₃ at pH 7.3. The relevant fractions were collected andpooled into a single PGAG sample and lyophilized in preparation fordesalting.

The PGAG sample was desalted through four sequentially joined PharmaciaHiPrep™ 26/10 (17-5087-01) columns at a flow rate of 10 ml/min on aBiologic DuoFlow system (Biorad) using a QuadTec UV-Vis detector. Therelevant fractions were collected and pooled into a single sample setand lyophilized in preparation for further treatment.

In the fourth step, the PGAG sample set obtained from the desaltingprocedure was subjected to a pronase and neuraminidase treatment, inorder to digest away core proteins and to subsequently liberate GAGchains. In this respect, lyophilized PGAG samples were resuspended in aminimum volume of 25 mM sodium acetate (pH 5.0) and clarified byfiltration through a 0.4 μm syringe filter. The total sample volume wasdispensed into 10 ml glass tubes in 500 μl aliquots. To this aliquot wasadded 500 μl of 1 mg/ml neuraminidase before the mixture was incubatedfor 4 hours at 37° C. Following incubation, 5 ml of 100 mM Tris-acetate(pH 8.0) was added to each sample. An additional 1.2 ml of 10 mg/mlpronase, reconstituted in 500 mM Tris-acetate and 50 mM calcium acetate(pH 8.0), was added to each sample before the mixture was incubated for24 hrs at 36° C. Following this treatment, all volumes were combined andprepared for anion exchange chromatography by centrifugation andfiltration.

In a fifth step, the GAG sample isolated following protein cleavage waseluted through a Pharmacia XK 26 (56-1053-34) column packed with Capto QAnion Exchange Beads (Biorad) at a flow rate of 5 ml/min on a BiologicDuoFlow system (Biorad) using a QuadTec UV-Vis detector. In thisrespect, the sample was loaded in a low salt buffer containing PBS (150mM NaCl w/o Ca²⁺ and Mg²⁺) and 0.02% NaN₃ at pH 7.3. The sample waseluted in a high salt buffer containing PBS (150 mM NaCl w/o Ca²⁺ andMg²⁺), 850 mM NaCl and 0.02% NaN₃ at pH 7.3. The relevant fractions werepooled, lyophilized and desalted as per the aforementioned protocol fordesalting the PGAG sample.

N-terminal biotinylated peptide (1 mg), corresponding to theheparin-binding domain of BMP-2, and comprising an amino acid sequencerepresented by QAKHKQRKRLKSSCKRH [SEQ ID NO:6], was mixed with low saltbuffer containing PBS (150 mM NaCl w/o Ca²⁺ and Mg²⁺). The mixture waseluted through a column packed with a streptavidin-coated resin matrix.The column was then exposed to a high salt buffer containing PBS (150 mMNaCl w/o Ca²⁺ and Mg²⁺), 850 mM NaCl and 0.02% NaN₃ at pH 7.3, toascertain whether, under those conditions the peptide had bound securelyto the matrix. No substantial loss of peptide from the column wasobserved. The column was subsequently washed with the low salt buffer inpreparation for sample loading.

The GAG mixture (2 mg), isolated using the procedure outlined in Example1, was suspended in low salt sodium phosphate buffer (1 mL), and loadedonto the peptide column of Example 2. The sample was eluted with a lowsalt buffer containing PBS (150 mM NaCl w/o Ca²⁺ and Mg²⁺). A peakcorresponding to GAGs with negligible BMP-2 affinity was observed in theUV-Vis detector trace. The column fractions responsible for giving riseto this peak were combined. These fractions are known as ‘GAG-’—theminus sign denoting the lack of affinity with the column. When it becameevident from the UV-Vis detector that the trace had flattened to thebaseline, and that no further oligosaccharide was eluting, the elutingsolvent was changed to a high salt buffer containing PBS (150 mM NaClw/o Ca²⁺ and Mg²⁺), 850 mM NaCl and 0.02% NaN₃ at pH 7.3. Following thischange in the eluting solvent, a peak corresponding to BMP-2 specificGAGs was observed in the UV-Vis detector trace. The column fractionsresponsible for giving rise to this peak were combined. These fractionsare known as ‘GAG+’—the plus sign denoting the presence of affinity withthe column. In the case of GAG compounds sourced from preosteoblastcells, the GAG+ fraction represented 10% of the overall GAG mixture.

Example 5

The addition of BMP2 has a clearly defined capacity to induce osteogenicdifferentiation in C2C12 myoblasts. Similarly, the pre-incubation ofBMP2 with heparin has been shown to both extend the cytokines half lifeand its immediate potency in vitro. Here we examined the capacity ofGAG+ and GAG− fractions to augment the osteoinduction of C2C12 cells invitro by BMP2.

The GAG+ sample from Example 4 (0, 10, 100, 1000 ng/mL) was added toC2C12 myoblasts in vitro in the presence of BMP-2 (0, 50, 100 ng/mL).Measurement of the relative expression of the osteocalcin gene indicatedthat the GAG+ sample was able to potentiate BMP-2 to effect osteocalcingene expression at levels of BMP-2 far below those currently used intherapy (300 ng/mL). The results of this assay (including calculatedp-values and errors) are represented graphically in FIG. 34 in which theexperimental conditions for each ‘culture condition’ are as follows:

-   -   1. Control cells, no BMP-2 added, no GAG added    -   2. BMP-2 at 50 ng/mL    -   3. BMP-2 at 50 ng/mL, GAG+ at 10 ng/mL    -   4. BMP-2 at 50 ng/mL, GAG+ at 100 ng/mL    -   5. BMP-2 at 50 ng/mL, GAG+ at 1000 ng/mL    -   6. BMP-2 at 100 ng/mL    -   7. BMP-2 at 100 ng/mL, GAG+ at 10 ng/mL    -   8. BMP-2 at 100 ng/mL, GAG+ at 100 ng/mL    -   9. BMP-2 at 100 ng/mL, GAG+ at 1000 ng/mL

Interestingly, while 1000 ng/ml of GAG+ is able to significantly augmentBMP2 mediated osteocalcin expression, the addition of concentrations ofGAG+ below 1000 ng/ml appear to progressively inhibit this expression.Furthermore, the addition of sufficient GAG+ also managed to drive theinduction of osteocalcin by 50 ng/ml of BMP2 above that of 100 ng/ml ofBMP2 on its own, indicating the potency of this interaction.

This cell culture based analysis demonstrated that the addition of GAG+to C2C12 osteogenic cultures together with BMP2 resulted in asignificant upregulation of osteocalcin expression indicating anincrease in BMP2 signalling efficacy. This result supports the specificassociation of GAG+ chains with BMP2, thereby blocking the BMP2-HBP andpreventing its association with matrix-based PGAGs. The resultingupregulation of osteogenic gene expression is comparable to thatobserved in previous studies utilising heparin to achieve a similareffect. Interestingly, the addition of concentrations of GAG+ that fallbelow 1000 ng/ml appear to have an initially antagonistic effect on BMP2signalling.

One possible hypothesis to explain this observation revolves around thecapacity for a given number of GAG+ molecules to bind a certain numberof BMP2 molecules. Under conditions where no exogenous GAG+ is added tothe culture system the majority of BMP2 molecules will be able toassociate with the ECM, thereby being localised away from their cognatereceptors and being unable to immediately initiate signalling.Subsequent dissociation of BMP2 from the ECM, both spontaneously and bytargeted enzymatic alteration of their associated GAG chains, has thecapacity to induce long term BMP2 signalling. The addition of a largenumber of GAG+ molecules to this system, as is the case in samplessupplemented with 1000 ng/ml of GAG+, permits the majority of BMP2molecules to remain in solution where they are free to mediate receptordimerisation and induce downstream signalling. Both these processes ofcytokine/receptor interaction likely require particular concentrationthresholds in order maintain an efficient level of signalling. Underculture conditions containing 50 ng/ml of BMP2, the addition of lowconcentrations of GAG+ allows for a portion of the available cytokine toremain soluble while the remaining portion associates with the ECM.Under these conditions only a small quantity of BMP2 remains solublebut, due to its low concentration, becomes highly diffuse in the medialeading to negligible signalling. Similarly, due to a portion of theBMP2 remaining solubilised, a reduced quantity of BMP2 can be found inthe ECM, resulting in a decrease in signalling from BMP2 liberated fromthe ECM by direct cellular activity. However, under culture conditionscontaining 100 ng/ml of BMP2 the combined effects of soluble and ECMbased BMP2 are, with the addition of 100 ng/ml of GAG+, sufficient toinduce BMP2 signalling similar to control levels. Without further study,however, the dynamics involved in BMP2/GAG+ signalling remain unclear.Future studies utilising surface plasmon resonance may help elucidatethe efficiency of BMP2/GAG+ interactions and may aid in clarifying theseobservations.

Example 6

The enzyme heparanase 3 was used to cleave GAG+ and GAG− sugar chainsfrom Example 4 according to the following method. GAG+ and GAG− wereeach treated separately at a concentration of 4 mg/mL, with heparanase 3(250 mU enzyme per 100 μg oligosaccharide) for 16 hours at 37° C.Subsequently, the mixture was heated for 5 minutes at 70° C. toinactivate the heparanase 3. The digested GAG+ and GAG-mixtures wereeach subjected to the peptide column separately. The UV-Vis detectortrace of each chromatographic run indicated that the digested materialshowed the same affinity for the column as the undigested material.

Example 7 Coupling of Biotinylated Peptide to Streptavidin Column

Method: BMP2 HB—peptide was dissolved in 20 mM phosphate buffer, 150 mMNaCl (Low Salt Buffer), at a concentration of 1 mg/ml. The peptidesolution was subjected to affinity chromatography on a streptavidincolumn (1 ml) equilibrated in low salt buffer using a low-pressureliquid chromatography (Biologic-Duoflow chromatography system fromBio-Rad). The medium was loaded at a flow rate of 0.2 ml/min and thecolumn washed with the same buffer until the baseline reached zero. Tocheck that the peptide actually attached to the column, the column waseluted with a step gradient of 1.5 M NaCl (high salt buffer) andre-equilibrated with low salt buffer.

The BMP2 heparin binding site (5 mg) sequence (QAKHKQRKRLKSSCKRHP-NHETbiotin (SEQ ID NO: 1)) was synthesized and coupled to the 1 mlstreptavidin column (GE Healthcare). The chromatogram (FIG. 36) showsall peptides bounds tightly to the streptavidin beads.

Purification of BMP2-Specific Heparan Sulfate

Method: Celsus HS was dissolved in 20 mM phosphate buffer, 150 mM NaCl(Low Salt Buffer), at a concentration of 1 mg/ml. The peptide solutionwas subjected to affinity chromatography on a streptavidin column (1 ml)equilibrated in low salt buffer using a low-pressure liquidchromatography (Biologic-Duoflow chromatography system from Bio-Rad).The medium was loaded at a flow rate of 0.2 ml/min and the column washedwith the same buffer until the baseline reached zero. The bound BMP2specific HS was eluted with a step gradient of 1.5 M NaCl (high saltbuffer), the peak factions were collected, and the columnre-equilibrated with low salt buffer. The elution peak (BMP2+ve) andflow through peak (BMP2−ve) HS were collected separately, freeze-driedand stored at −20° C.

The chromatogram (FIG. 37) shows a small portion (˜15-20%) of the HSspecifically bound to the column and that it eluted in the high saltbuffer.

Desalting of BMP2 Peptide Column Bound HS

Method: BMP2 specific HS was dissolved in 10 ml distilled water. Thesamples were subjected to desalting chromatography on a Hi-prepdesalting column (10 ml) equilibrated in distilled water using alow-pressure liquid chromatography (Biologic-Duoflow chromatographysystem from Bio-Rad). The HS was loaded at a flow rate of 10 ml/min andthe column washed with distilled water. The pure HS fractions werecollected, freeze-dried and stored at −20° C.

The chromatogram (FIG. 38) shows a clear separation of pure BMP2specific HS (absorbance peak) and the Salt buffer (conductance peak).

Desalting of BMP2 Peptide Column Unbound HS

Method: The non-specific HS was dissolved in 10 ml distilled water. Thesamples were subjected to desalting chromatography on a Hi-prepdesalting column (10 ml) equilibrated in distilled water using alow-pressure liquid chromatography (Biologic-Duoflow chromatographysystem from Bio-Rad). The HS was loaded at a flow rate of 10 ml/min andthe column washed with distilled water. The pure HS fractions werecollected, freeze-dried and stored at −20° C.

The chromatogram (FIG. 39) shows a clear separation of the unbound HS(absorbance peak) and the Salt buffer (conductance peak).

SAX-HPLC Disaccharide Analysis—BMP2 Positive HS

Method: Samples (100 μg) were dissolved in 100 mM sodium acetate/0.2 Mcalcium acetate, pH 7.0. Heparinase, heparitinase I and II were all usedat a concentration of 10 mU/ml in the same buffer. Each sample wassequentially digested for a recovery of disaccharides for SAX-HPLCanalysis; for this the samples were digested at 37° C. as follows:heparinase for 2 h, heparitinase I for 1 h, heparitinase II for 18 h,and finally an aliquot of each lyases for 6 h. Samples were run on aBioGel P-2 column (1×120 cm) equilibrated with 0.25 M NH₄HCO₃. Thedisaccharide peak was lyophilized and then dissolved in acidified water(pH 3.5 with HCl). This was passed over a ProPac PA-1 SAX-HPLC column(Dionex, USA), attached to a high pressure liquid chromatography systemand the HS disaccharides eluted with a linear gradient 0 to 1.0 M NaCl,pH 3.5, over 60 min at a flow-rate of 1 ml/min. The peaks identifiedusing HS disaccharides standards (Seikagaku, Tokyo, Japan and Iduron)monitored at A₂₃₂ nm.

The HS retained by the BMP2 peptide affinity column was subjected to anenzymatic disaccharide analysis by exposing it to a combination ofheparin lyases (heparinase, heparitinase I and II) to completion andthen subjecting the resulting disaccharide fragments to strong anionexchange HPLC (SAX-HPLC). The peaks on the chromatogram (FIG. 40) allowus to estimate the relative proportions of each of the componentdisaccharides within the binding HS population. The analysis shows thatgreater proportion of disaccharides in the BMP2-binding peptide HSpopulation have an N-sulfated glucosamine.

SAX-HPLC Disaccharide Analysis—BMP2 Negative HS

The HS that did not the BMP2 peptide affinity column was subjected to anenzymatic disaccharide analysis by exposing it to a combination ofheparin lyases (heparinase, heparitinase I and II) to completion andthen subjecting the resulting disaccharide fragments to strong anionexchange HPLC (SAX-HPLC). The peaks on the chromatogram (FIG. 41) allowus to estimate the relative proportions of each of the componentdisaccharides within the HS population. The analysis shows that greaterproportion of disaccharides in the flow through HS population have anN-sulfated glucosamine.

SAX-HPLC Disaccharide Profile of Celsus Total HS

The Total HS bought from Celsus as the starting material was alsosubjected to a enzymatic disaccharide analysis by exposing it to acombination of heparin lyases (heparinase, heparitinase I and II) tocompletion and then subjecting the resulting disaccharide fragments tostrong anion exchange HPLC (SAX-HPLC). The peaks on the chromatogram(FIG. 42) allow us to estimate the relative proportions of each of thecomponent disaccharides within the HS population.

ESPR—Analysis of BMP2 Positive and BMP2 Negative HS

Method: BMP2+ve and −ve HS (10 mg/ml) was dissolved in 1 ml 0.1 M MES,pH 5.5 and 300 μl 0.1 MES, pH 5.5, containing 2 mg/mlbiotin-LC-hydrazide (Pierce), EDC (7 mg) was added to the mixture andincubated at room temperature for 2 h before addition of another 7 mg ofEDC. After a further 2 h incubation, unincorporated biotin was removedwith a desalting column (Amersham Pharmacia). The BMP2+ve and −ve HSwere tested for their capacity to bind soluble BMP2. Real time bindinganalysis was carried out using SPR, wherein biotin thiol-coated goldsensor chips were used as a platform for immobilized streptavidin. Usinga biotin-streptavidin-biotin bridge, biotinylated HS could beimmobilized on the sensor chip. The growth factor (200 nM) was thenadded to the solution bathing the immobilized HS and incubated for 20min. Real time binding was monitored by measuring the change in theminimum reflectance angle (θ) over time.

FIG. 44 shows Surface Plasmon Resonance (SPR) analysis of protein-sugarinteractions. As shown by the curves, which reflect the avidity of the“on-rate” (Ka), BMP2 does not bind as avidly to the “flow-through” HS,as evidenced by the smaller angle shift, as to the BMP2-binding HS.

BMP2 Binding Capacity of BMP2+ve and BMP2 −ve Celsus HS PreparationsCoated on an Iduron Heparin/GAG Binding Plate

Method: BMP2 was dissolved in Blocking Solution (0.2% gelatin in SAB) ata concentration of 3 μg/ml and a dilution series from 0-3 μg/ml inBlocking Solution established. Dispensing of 200 μl of each dilution ofBMP2 into triplicate wells of Heparin/GAG Binding Plates pre-coated withheparin; incubated for 2 hrs at 37° C., washed carefully three timeswith SAB and 200 μl of 250 ng/ml biotinylated anti-BMP2 added inBlocking Solution. Incubation for one hour at 37° C., wash carefullythree times with SAB, 200 μl of 220 ng/ml ExtrAvidin-AP added inBlocking Solution, Incubation for 30 mins at 37° C., careful washingthree times with SAB and tap to remove residual liquid, 200 μl ofDevelopment Reagent (SigmaFAST p-Nitrophenyl phosphate) added.Incubation at room temperature for 40 minutes with reading at 405 nmwithin one hour.

The specially-prepared plate surface (Iduron) adsorbs GAGs withoutmodification whilst retaining their protein binding characteristics.Binding occurs at room temperature from physiological buffers. Theresults (FIG. 45) demonstrate the greater affinity of the BMP2-selectedHS preparations for BMP2 over the flow-through or native preparations.BMP2 acted as the control.

ALP Activity of BMP2 Positive and Negative HS on C2C12 Cells

Methods: ALP Assay. C2C12 cells were plated at 20,000 cells/cm² in a24-well plate in DMEM (Sigma-Aldrich Inc., St. Louis, Mo.) containing10% FCS (Lonza Group Ltd., Switzerland) and antibiotics (1% Penicillinand 1% Streptomycin) (Sigma-Aldrich Inc., St. Louis, Mo.) at 37° C./5%CO₂. After 24 hours, the culture media was switched to 5% FCS low serummedia containing different combinations of 100 ng/mL BMP2 (R&D Systems,Minneapolis, Minn.), 3 mg/mL Celsus HS and varying concentrations ofBMP2-specific (+ve HS) and non-specific (−ve HS) Celsus HS preparations.Cell lysis was carried out after 3 days using RIPA buffer containing 1%Triton X-100, 150 mM NaCl, 10 mM Tris pH 7.4, 2 mM EDTA, 0.5% Igepal(NP40), 0.1% Sodium dodecyl sulphate (SDS) and 1% Protease InhibitorCocktail Set III (Calbiochem,Germany). The protein content of the celllysate was determined by using BCA protein assay kit (Pierce ChemicalCo., Rockford, Ill.). ALP activity in the cell lysates was thendetermined by incubating the cell lysates with p-nitrophenylphosphatesubstrate (Invitrogen, Carlsbad, Calif.). The reading was normalized tototal protein amount and presented as relative amount to the groupcontaining BMP2 treatment alone.

FIG. 46 shows BMP-2 specific HS (+ve HS) enhanced alkaline phosphatase(ALP) activity induced by BMP-2 at a greater degree compared tonon-specific HS (−ve HS). BMP-2 at 100 ng/mL was introduced alone or incombination with 30 μg/mL Celsus HS or varying concentration of specificand non-specific HS. Specific and non-specific HS was introduced aloneat 30 μg/mL.

ALP Staining

Method: ALP Staining. C2C12 cells were cultured as described above.After 3 days of treatment, the cell layer was washed in PBS and stainedusing Leukocyte Alkaline Phosphatase Kit (Sigma-Aldrich Inc., St. Louis,Mo.) according to manufacturer's specification. Briefly, the cell layerwas fixed in citrate buffered 60% acetone and stained in alkaline-dyemixture containing Naphthol AS-MX Phosphatase Alkaline and diazoniumsalt. Nuclear staining was performed using Mayer's Hematoxylin solution.

BMP-2 specific HS (+ve HS) enhanced alkaline phosphatase (ALP) activityinduced by BMP-2 at a greater degree compared to non-specific HS (−ve)when evaluated histochemically (FIG. 47). BMP-2 at 100 ng/mL wasintroduced in combination with 0, 0.3, 3 and 30 μg/mL of GAG.

BMP2 Stability

Method: Smad 1/5/8 Phosphorylation. C2C12 cells were plated at 20,000cells/cm² in a 24-well plate in DMEM (Sigma-Aldrich Inc., St. Louis,Mo.) containing 10% FCS (Lonza Group Ltd., Switzerland) and antibiotics(1% Penicillin and 1% Streptomycin) (Sigma-Aldrich Inc., St. Louis, Mo.)at 37° C./5% CO2. After 24 hours, the culture media was switched to 5%FCS low serum media. Treatment conditions containing 100 ng/mL BMP2 (R&DSystems, Minneapolis, Minn.) in the presence/absence of 3 mg/mL ofheparin (Sigma-Aldrich Inc., St. Louis, Mo.) or BMP2-specific (+ve HS)Celsus HS were added 24 hours after the cells have been equilibrated inlow serum media. Cell lysate was harvested in 1× Laemmli buffer at 0,24, 48 and 72 hour time points. The lysate was separated in NuPAGE Novex4-12% Bis-Tris Gel (Invitrogen, Carlsbad, Calif.) and analyzed withwestern blot using antibodies against Phospho-Smad 1/5/8 (CellSignaling, Danvers, Mass.) and Smad 1/5/8 (Santa Cruz BiotechnologyInc., Santa Cruz, Calif.).

The ability of the BMP2-binding HS, i.e. HS3, to prolong the effects ofBMP2 on cells (presumably in part by protecting the protein againstproteolytic degradation) was compared to the effects of commercialheparin. C2C12 cells were exposed to nothing, BMP2 alone, BMP2+Heparinor BMP2+HS3 for 72 hours and the levels of phosphorylation of theBMP2-specific intracellular signaling molecule Smad1/5/8 monitored byimmunoblotting (FIGS. 55 and 84). The results demonstrate that the HS3can prolong BMP2 signalling to levels that equal or exceed those ofheparin.

Example 8

This experiment was designed to investigate whether HS3, when combinedwith Smith & Nephew's bone void filler JAX™ gel+Tri-calcium phosphate(TCP) stars can speed up long bone repair.

A non union critical defect is created in the ulna of adult rabbits, thestars placed in the defect, the wound closed and repair monitored after4 and 8 weeks with a combination of histology and imaging.

Biomaterials

JAX™ is a 3-tricalcium phosphate (TCP) synthetic bone substitutemanufactured by Smith and Nephew Orthopaedics Ltd, USA. JAX™ consists ofsix-armed granules, which interlock to provide 55% intergranularporosity in a defect site, allowing cell and vascular infiltration. Theclinical indication is for non-load bearing bony defects of 4-5 cm. JAX™also includes a hydrogel component.

In Vitro Study

The release of heparin at either high or low concentrations (as asubstitute for HS) from Jax gel/TCP mixtures in vitro was assessed byfirst labeling it with Alexa Fluor 488 dye and then monitoring itsrelease into PBS in culture plates. Release in both cases was rapid andbursting.

Fluorescent Labeling of Heparin.

For non-biological in vitro assays, heparin, a hypersulfated member ofthe HS glycosaminoglycan family, was conjugated with Alexa Fluor 488(A488, Molecular Probes, UK) using a method published previously by ourgroup (E. V. Luong, L. Grondahl, V. Nurcombe, S. Cool. In vitrobiocompatibility and bioactivity of microencapsulated heparan sulfateBiomaterials 2007; 28:2127 2136). Briefly, 3 mg of heparin (H-3149) wassolubilized in 300 μL of 0.1 M solution of 4-morpholinoethanesulfonicacid (MES, M3671) buffer (pH 4.5) and combined with 50 μL of a 10%1-ethyl-3-(3-dimethylaminopropryl) carbodiimide hydrochloride (EDC,Fluke 03449) solution in 0.1 M MES buffer. Subsequently, a 1% A488solution (50 μl) in 0.1 M MES buffer was added to the heparin/EDCsolution. The mixture was protected from light and incubated overnightat room temperature. The fluorescently conjugated heparin was eluted onan Amersham PD10 desalting column. The labeling efficiency wasapproximately 1.3 mol A488/mol heparin.

Release Profile.

Three JAX™ granules were loaded with either 17 or 170 μg of A488-heparin(50 μL in 100 μL hydrogel), protected from light, and placed in 1 mL ofPBS at 37° C. for 48 h. At 1, 2, 3, 4, 5, 6, 24 and 48 h, 100 μL ofconditioned phosphate buffered saline (PBS) was collected for samplingand replaced with fresh PBS. The concentration of released A488-heparinwas quantified by fluorometry and cumulative release of A488-heparin wasreported as a percentage of loading concentration (FIG. 57).

In Vivo Study Experimental Design.

See FIG. 56) Twenty male New Zealand White rabbits (weighing 2-2.5 kg)received bilateral ulna defects. Each defect was randomly assigned toone of three experimental groups. Every defect received 18 JAX™ granulesand 150 μL of hydrogel containing one of the following treatments: 30 μgHS, 100 μg HS or an equal volume of PBS (50 μL). After 4 and 8 weeks ofimplantation, the rabbits were sacrificed and ulnas were harvested. Fourulnas per treatment were assessed non-destructively using 2D X-rays andmicro-computed tomography (micro-CT) for mineral formation at bothtime-points. Subsequently, these ulnas were processed for histology andimmunohistochemistry. At week 8, an additional three samples pertreatment were included for evaluation by torsional testing. Somedefects were left empty to serve as internal controls to ensure that themodel Was truly non-union.

X-Ray Monitoring of New Bone Formation in Defect Sites

HS3 was applied in Jax gel at two different concentrations (30 and 100ug—called HS30 and HS100) and assessed for new bone formation comparedto no treatment over 0, 4 and 8 weeks. The HS-treated animals show clearindication of new bone formation over the controls.

Radiographic Analyses.

JAX™ granules are radio-opaque and therefore difficult to distinguishfrom new bone in the defect site on 2D x-rays. However, at the earlytime points, voids between the granules and immediately adjacent to theradius are clearly visible and the progression of bone formation inthese spaces can be monitored (S. A. Clarke, N. L. Hoskins, G. R.Jordan, D. R. Marsh. Healing of an ulnar defect using a proprietary TCPbone graft substitute, JAX™, in association with autologous osteogeniccells and growth factors. Bone 2007; 40: 939-947). An ImagingRadiographic System (MUX-100, Shimadzu) was used to capture 2D images ofthe ulna defects immediately after the surgery and at weeks 4 and 8.Digital micrographs are then taken of the X-rays. X-rays were takenunder general anesthesia. X-ray micrographs are shown in FIGS. 52, 53and 58.

Micro CT Monitoring of New Bone Formation in Defect Sites

HS3 given at doses of 30 and 100 ug (HS30 and HS100) at the time ofsurgery was compared to PBS star alone controls using micro CT(computerized tomography) imaging (FIGS. 52, 53 and 59).

Micro-CT Analyses.

At weeks 4 and 8, harvested ulnas were scanned with a micro CT scanner(Skyscan 1076; Skyscan, Belgium). Scanning was performed with aresolution of 35 μm and a scanning width of 68 mm. The scanner was setat a voltage of 104 kV and a current of 98 μA. Cone-BeamCT-reconstruction A Sasov software (Skyscan) was used to convert theisotropic slice data obtained into 2D images. For this reconstruction,the lower and upper threshold values for bone were assumed to be −315and 543 Hounsfield units. The data was then analyzed and remodeled usingthe associated CTAn software (Skyscan) for quantification and Mimics11.1 software (Materialise, Belgium) to render 3D images. A cylindricalregion of interest (ROI, cocentrically positioned over the defect site)and the total number of slices (corresponding to the length of thedefect) was kept constant for all the samples. The total volume of newlyformed bone within the ROI was measured by assigning predeterminedthresholds for total bone content, cortical bone (JAX™ and radii) andtrabecular bone (or newly formed bone). The data was reported as bonevolume/total volume (%).

HS3 (at both 30 μg and 100 μg doses) significantly increased the BV/TV(%) as compared to controls. There was no significant difference betweenHS30— and HS100-treated ulnas (FIG. 60).

Histology

After the designated experimental periods, bone was harvested, fixed,decalcified, sectioned and mounted for staining with various dyes.

FIGS. 61 and 62 show H&E staining (vide infra) for the 3 treatmentgroups over weeks 4 and 8. HS3-treatment clearly shows more tissueinfiltrating the defect than in controls.

Higher magnification H&E-stained micrographs (FIG. 62) revealed new bonebeing deposited immediately adjacent to the Jax stars (the clearerislands), with greater amounts of bone, bone marrow and cartilaginoustissue apparent in the HS treated animals. By week 8, in the HS-treatedulnas, new bone has remodeled and matured.

Histological Analyses.

The extracted ulnas were fixed in 10% neutral buffered formalin for 1week under vacuum, and decalcified in 15% EDTA, pH 7.2, for 4 weeks atroom temperature. Then, the ulnas were processed using a vacuuminfiltration processor (Sakura Finetek, Japan) with a 14 h program.After dehydration and clearing, the bones were embedded in Paraplastparaffin wax (Thermo Scientific) and the paraffin blocks sectionedlongitudinally at 5 μM using a rotary microtome (Leica Microsystems,Germany). Paraffin sections were placed on positively charge microscopeslides, dried, stained with Hematoxylin/Eosin and Modified Tetrachromeand finally examined under an Olympus Stereo (SZX12) and uprightfluorescence microscope (BX51).

FIGS. 63 and 64 show Ralis Tetrachrome (Z. A. Ralis, G. Watkins.Modified tetrachrome method for osteoid and defectively mineralized bonein paraffin sections. Biotech and Histochem 1992; 67: 339-345) staining(vide infra) for the 3 treatment groups over weeks 4 and 8. HS-treateddefects clearly show more tissue infiltrating the defect than incontrols.

Higher magnification Ralis Tetrachrome-stained micrographs (FIG. 64)revealed new bone being deposited immediately adjacent to the Jax stars(the clearer islands), with greater amounts of woven bone, bone marrowand capillaries apparent in the HS3 treated animals. By week 8, in theHS-treated ulnas, new bone has remodeled and matured.

Immunostaining

After the designated experimental periods, bone was harvested, fixed,decalcified, sectioned and mounted for staining with various dyes. FIG.65 shows immunostaining for the late osteogenic marker osteocalcin (videinfra) for the 3 treatment groups over weeks 4 and 8. HS3-treatedspecimens clearly show more positive (brown) staining filling up thedefect than in controls.

Higher magnification (FIG. 66) of the osteocalcin staining revealed newbone being deposited immediately adjacent to the Jax stars (the clearerislands), with greater amounts of remodelling cavities that consist ofbone marrow, capillaries and osteoblast-lined borders.

Immunohistochemistry Analysis.

Deparaffinised sections were washed with PBS, incubated with ProteaseXXIV (BioGenex, San Ramon, USA) for 10 min for antigen retrieval,followed by incubation with 0.3% hydrogen peroxide in water for 20 minat room temperature. After washing, sections were blocked with 5% normalgoat serum in PBS for 30 min. Tissue sections were incubated withappropriate concentrations of primary antibodies: osteocalcin (ab13420,1:150, Abcam, UK) or the same concentration of mouse IgG (MG100, CaltageLab, USA; as negative controls) in blocking buffer overnight at 4° C.Sections were washed three times with PBS, and then incubated with ratabsorbed biotin-labeled anti-mouse IgG (Vector Lab Inc, USA) for 1 h.Sections were washed with PBS and incubated with avidin-biotinperoxidasecomplex (ABC) solution (Immunopure ABC preoxidase staining kit, VectorLab. Inc) for 1 h. Peroxidase activity was detected using3,3-diaminobenzidiine-tetrahydrochloride (DAB; DAKO, USA). Sections werewashed, mounted and examined under bright field microscopy using anOlympus SZX12 stereomicroscope.

Torsional Testing

After the designated experimental periods, bones were tested for theirmechanical strength. FIG. 67 shows the torsional testing set-ups.

Torsional Testing. After sacrifice at 8 weeks post-surgery, the rabbitulnas were retrieved, wrapped in PBS-soaked gauze to maintain moisture,and frozen at −20° C. until torsional testing. Upon thawing, the ulnaends were potted in polymethylmethacrylate (Meliodent Rapid Repair,Heraeus Kulzer), contained within customized plastic blocks and allowedto solidify, to enable stable fixation. Ulnas were subsequently mountedin a MTS 858 Mini Bionix II testing system (MTS, Eden Prairie, Minn.).Polymer blocks and gauze were gently removed prior to testing. Eachspecimen was then tested to failure in torsion and the resultingtorque—angular displacement curves were recorded. The rotation rate usedwas 1 degree per second until 35 degrees was reached and data werecollected at 100 Hz. The stiffness, maximum torque, and angle at failurewere recorded for each specimen, with the stiffness being measured asthe slope of the linear portion of the torque—angular displacement curve(M. Bostrom, J. M. Lane, E. Tomin, M. Browne, W. Berberian, T. Turek, J.Smith, J. Wozney, T. Schildhauer. Use of bone morphogenetic protein-2 inthe rabbit ulnar non-union model. Clin Orthop Relat Res 1996; 327:272-282).

Statistical Analyses.

Quantitative data was obtained in triplicates and reported asmeans±standard deviation. Statistical analyses were performed using theStudent's t-test (GraphPad software), and a p-value of less than 0.05was considered significant.

Quantification of Stiffness and Maximum Torque Assessed from Control andHS-Treated Ulnae.

Stiffness was markedly improved for the HS-treated bones. As the starsoccupy the largest proportion of the defect, which became a physicalbarrier for new bone infiltration, it resulted in improvements that wereonly marked (as opposed to significant)—see FIG. 68.

Example 9 Evaluating Bone Regeneration in a Critical Sized DefectInduced by HS3-Loaded Collagen Sponges

The same overall approach to that of Example 8 was used in a secondstudy, except that the Jax TCP stars were replaced with FDA-approvedcollagen sponges.

Biomaterials.

Collagen sponges were purchased from Integra Life Sciences (HELISTAT,Integra Life Sciences Corp, USA) and measured 7×21×5 mm. These spongeswere processed from bovine deep flexor tendon, are bioabsorbable andnon-pyrogenic.

The morphology of the sponges was evaluated using Scanning ElectronMicroscopy (SEM). Briefly, collagen sponges were sputtered-coated withgold and then examined using SEM (Jeol JSM 5310 LV) at an acceleratingvoltage of 10 kV.

Biomolecules.

Heparan sulfates (HS) tested in this study were bone-morphogeneticprotein (BMP) specific HS, also known as HS3.

In Vivo Study

The study used HS3 loaded at 30 μg per sponge (HS30) and BMP-2 loaded at10 μg per sponge (BMP2-10). Bilateral ulna defects were created andtreated with HS30, BMP2-10, or HS30+BMP2-10, or PBS controls.

HS3 (30 μg) was applied in collagen sponges either alone or incombination with BMP2 (10 μg) and assessed for new bone formationcompared to no treatment over 0, 4 and 8 weeks. These were assessedagainst the negative collagen sponge control, and the positive BMP2control.

Experimental Design.

Twenty male New Zealand White rabbits (weighing 2-2.5 kg) receivedbilateral ulna defects. Each defect was randomly assigned to one ofthree experimental groups. Every defect received 1 collagen spongesoaked with one of the following treatments (total 300 μL, in PBS): 30μg HS, 10 μg BMP-2 (sometimes called BMP10), 30 μg HS+10 μg BMP-2 or anequal volume of PBS. After 4 and 8 weeks of implantation, the rabbitswere sacrificed and ulnas were harvested. Four ulnas per treatment wereassessed non-destructively using 2D X-rays and micro-computed tomography(micro-CT) for mineral formation at both time-points. Subsequently,these ulnas were processed for histology and immunohistochemistry. Atweek 8, an additional three samples per treatment were included forevaluation by torsional testing. Some defects were left empty to serveas internal controls to ensure that the model was truly non-union.

Surgical Procedures.

The research protocol for performing bilateral ulna osteotomies inrabbits was approved by the Institutional Animal Care and Use Committee,following all appropriate guidelines. All surgical procedures werecarried out under general anesthesia and aseptic conditions. Anesthesiaconsisted of a combination of ketamine (75 mg/kg) and xylazine (10mg/kg) injections as well as isoflurane via an induction chamber andfacemask for maintenance. A 6 cm skin incision was made and theoverlying muscle layers were parted until the length of the ulna wasexposed. A 1.5 cm longitudinal defect in the central diaphysis wascreated using an Acculan.

Radiographic Analyses.

An Imaging Radiographic System (MUX-100, Shimadzu, Japan) was used tocapture 2D images of the ulna defects immediately after the surgery andat weeks 4 and 8. Digital micrographs (FIG. 69-70) are then taken of theX-rays. X-rays were taken under general anesthesia. The collagen spongeswere not radio-opaque; hence it was easy to identify new bone in thedefect site on the 2D X-rays.

X-ray monitoring after 4 weeks reveals significant new bone filling thedefect sites in all the treatment cases as compared to the negativecontrols (FIG. 70).

X-ray monitoring after 8 weeks reveals the achievement of bone union forall treatments, but not in the negative control (FIG. 71).Interestingly, delivery of the HS3 alone resulted in bone union just asgood as that seen for the BMP2; the HS3 in combination with the BMP2 didnot accelerate this effect because it was already maximal.

Micro-CT Analyses.

At weeks 4 and 8, harvested ulnas were scanned with a pCT scanner(Skyscan 1076; Skyscan, Belgium). Scanning was performed with aresolution of 35 μm and a scanning width of 68 mm. The scanner was setat a voltage of 104 kV and a current of 98 μA. Cone-BeamCT-reconstruction A Sasov software (Skyscan) was used to convert theisotropic slice data obtained into 2D images. For this reconstruction,the lower and upper threshold values for bone were assumed to be −315and 543 Hounsfield units. The data was then analyzed and remodeled usingthe associated CTAn software (Skyscan) for quantification and Mimics11.1 software (Materialise, Belgium) to render 3D images. A cylindricalregion of interest (ROI, cocentrically positioned over the defect site)and the total number of slices (corresponding to the length of thedefect) was kept constant for all the samples. The total volume of newlyformed bone within the ROI was measured by assigning predeterminedthresholds for total bone content, cortical bone (radii) and trabecularbone (or newly formed bone). The data was reported as bone volume/totalvolume (%)—see FIG. 72.

Micro-CT Quantification of the percentage bone volume of total volume(BV/TV) for the treatment groups after weeks 4 and 8 confirmed theeffects of the HS3 alone were more than comparable with FDA-approvedBMP2 (FIG. 72).

Torsional Testing.

After sacrifice at 8 weeks post-surgery, the rabbit ulnas wereretrieved, wrapped in PBS-soaked gauze to maintain moisture, and frozenat −20° C. until torsional testing. Upon thawing, the ulna ends werepotted in polymethylmethacrylate (Meliodent Rapid Repair, HeraeusKulzer), contained within customized plastic blocks and allowed tosolidify, to enable stable fixation. Ulnas were subsequently mounted ina MTS 858 Mini Bionix II testing system (MTS, Eden Prairie, Minn.).Polymer blocks and gauze were gently removed prior to testing. Eachspecimen was then tested to failure in torsion and the resultingtorque—angular displacement curves were recorded. The rotation rate usedwas 1 degree per second until 35 degrees was reached and data werecollected at 100 Hz. The stiffness, maximum torque, and angle at failurewere recorded for each specimen, with the stiffness being measured asthe slope of the linear portion of the torque—angular displacement curve(M. Bostrom, J. M. Lane, E. Tomin, M. Browne, W. Berberian, T. Turek, J.Smith, J. Wozney, T. Schildhauer. Use of bone morphogenetic protein-2 inthe rabbit ulnar non-union model. Clin Orthop Relat Res 1996; 327:272-282).

Statistical Analyses.

Quantitative data was obtained in triplicates and reported asmeans±standard deviation. Statistical analyses were performed using theStudent's t-test (GraphPad software), and a p-value of less than 0.05was considered significant.

Quantification of Stiffness and Maximum Torque Assessed from Control,BMP2 and HS-Treated Ulnae.

Both stiffness and maximum torque was significantly improved for thetreatment groups. Remarkably, the HS3-alone treatment resulted inmechanical properties that were similar to BMP2 treatment and intactbone at week 8 (FIG. 73).

Example 10 Capillary Electrophoresis (CE) Analysis of Disaccharides

Heparan sulfate (HS) was from Celsus Laboratories Inc. (HO-03103, Lot#HO-10697). Disaccharide standards (ΔUA,2S-GlcNS,6S; ΔUA,2S-GlcNS,ΔUA,2S-GlcNAc,6S, ΔUA-GlcNS,6S, ΔUA-GlcNS, UA-GlcNAc, ΔUA,2S-GlcNAc, ΔUA-GlcNAc,6S, ΔUA,2S-GlcN, ΔUA,2S-GlcN,6S, ΔUA-GlcN,6S, ΔUA-GlcN Cat No.HD001 to HD013, Iduron Ltd, Manchester, UK), derived from the digestionof high-grade porcine heparin by bacterial heparinases, were purchasedfrom Iduron Ltd, Manchester, UK. A synthetic derivative of a notnaturally occurring disulfated disaccharide (ΔUA,2S-GlcNCOEt,6S), wasalso purchased from Iduron for use as an internal standard. HeparinOligosaccharides (dp4, dp6, dp8, dp10, dp12 (Cat. No. HO04, HO06, HO08,HO10, HO12)) and selectively desulfated heparin standards (2-0,6-O andN-desulfated heparin) (Cat No. DSH001/2, DSH002/6, DSH003/N, Iduron Ltd,Manchester, UK) were also purchased from Iduron Ltd, Manchester, UK.

Heparin lyase I (Heparitinase, EC 4.2.2.8, also known as heparitinaseI), heparin lyase II (heparitinase II, no EC number assigned) andheparin lyase III (heparinase, EC 4.2.2.7, also known as heparitinaseIII) were obtained from Seikagaku Corporation, Japan. The enzymes,supplied as lyophilised powders (0.1 U/vial), were dissolved in 0.1% BSAto give solutions containing 0.5 mU/μL. Aliquots (5 μL; 2.5 mU) werefrozen (−80° C.) until needed.

Digestion of HS Preparations with Heparin Lyase Enzymes

HS preparations (1 mg) were each dissolved in 500 μL of sodium acetatebuffer (100 mM containing 10 mM calcium acetate, pH 7.0) and 2.5 mU eachof the three enzymes was added. The samples were incubated at 37° C.overnight (24 h) with gentle inversion (9 rpm) of the tubes. A further2.5 mU each of the three enzymes was added to the samples which wereincubated at 37° C. for a further 48 h with gentle inversion (9 rpm) ofthe tubes. Digests were halted by heating (100° C., 5 min) and thenlyophilized. The digests were resuspended in 500 μL water and an aliquot(50 μL) was taken for analysis by CE.

Capillary Electrophoresis (CE)

The capillary electrophoresis operating buffer was made by adding anaqueous solution of 20 mM H₃PO₄ to a solution of 20 mM Na₂HPO₄.12H₂O togive pH 3.5. The column wash was 100 mM NaOH (diluted from 50% w/wNaOH). The operating buffer and column wash were both filtered using aMillipore filter unit fitted with 0.2 μm cellulose acetate membranefilters (47 mm ø; Schleicher and Schuell, Dassel, Germany).

Stock solutions of the 12 disaccharide standards were prepared bydissolving the disaccharides in water (1 mg/mL). To determine thecalibration curves for the standards, a mix containing all twelvestandards was prepared. The stock solution of the 12 standard mixcontained 10 μg/100 μL of each disaccharide and a dilution seriescontaining 10, 5, 2.5, 1.25, 0.625, 0.3125 μg/100 μL was prepared;including 2.5 μg of internal standard (ΔUA,2S-GlcNCOD,6S). The digestsof HS were diluted (50 μL/mL) with water and the same internal standardwas added (2.5 μg) to each sample. The solutions were freeze-dried andre-suspended in water (1 mL). The samples were filtered using PTFEhydrophilic disposable syringe filter units (0.2 μm; 13 mm Ø; Advantec,Toyo Roshi Kaisha, Ltd., Japan).

The analyses were performed using an Agilent^(3D)CE (AgilentTechnologies, Waldbronn, Germany) instrument on an uncoated fused silicacapillary tube (75 μm ID, 64.5 cm total and 56 cm effective length,Polymicro Technologies, Phoenix, Ariz., Part Number TSP075375) at 25° C.using 20 mM operating buffer with a capillary voltage of 30 kV. Thesamples were introduced to the capillary tube using hydrodynamicinjection (50 mbar×12 sec) at the cathodic (reverse polarity) end.Before each run, the capillary was flushed with 100 mM NaOH (2 min),with water (2 min) and pre-conditioned with operating buffer (5 min). Abuffer replenishment system replaced the buffer in the inlet and outlettubes to ensure consistent volumes, pH and ionic strength weremaintained. Water only blanks were run at both the beginning, middle andend of the sample sequence. Absorbance was monitored at 232 nm. All datawas stored in a ChemStore database and was subsequently retrieved andre-processed using ChemStation software.

Eleven of the 12 heparin disaccharides in the standard mix wereseparated using conditions detailed above. The 12th disaccharide,ΔUA-GlcN, does not migrate under the conditions used for theseexperiments. However, this disaccharide has not been reported to occurin heparan sulfates. The R2 values for the standard calibration curvesranged from 0.9949 to 1.0.

The heparin lyase I, II and III digest of the five HS preparations wasdone in duplicate and each duplicate was injected twice in the CE.Therefore, the normalized percentage of the disaccharides in the HSdigest is the mean average of the results for the four analyses. Of the11 disaccharides separated in the standard mixes, only eight of theseare detected in the HS digests. Other small signals are seen on thebaseline of the electrophoretograms of the digests and these maycorrespond to oligsaccharides >2 dp. As mentioned above, the largeroligosaccharides will have less UV absorbance compared with thedisaccharides.

The proportion of the eight disaccharides in the Celsus HS digests weresimilar to other previous analyses with a large component of ΔUA-GlcNAcand ΔUA-GlcNS and lesser proportions of ΔUA-GlcNAc,6S, ΔUA-GlcNS,6S andΔUA,2S-GlcNS,6S. This corresponds to the large proportion of mono- andunsulfated disaccharide lesser proportions of disulfated disaccharideand small proportion of trisulfated disaccharide consistent with theHPLC-SEC profiles. The non-retained HS is enriched in the mono- andun-sulfated disaccharides compared with the Celsus HS starting material(FIG. 76). This pattern for the non-retained material was also seenquite distinctly in HPLC-SEC chromatograms.

The distinctive feature of the disaccharide analysis of the HS3preparation is the enrichment of the trisulfated disaccharide anddepletion of the mono- and un-sulfated disaccharides (FIG. 76) comparedwith the Celsus HS starting material. The enrichment of the trisulfatedmaterial is also evident after HPLC-SEC. The disaccharide compositionfor the bulk HS3 preparation was similar to the disaccharide compositionof the HS3 from the initial run and the final run.

Whilst the tri-sulfated disaccharide species was present at asignificantly higher level in HS+ve compared to HS_(pm) or HS_(−ve),there was enhancement of only specific di-sulfated species, supportingthe view that sequence-specific moieties were responsible for the BMP-2affinity, rather than any simple overall charge-density effect.

In conclusion, the disaccharide data appears self-consistent in that anincrease in the relative proportion of one disaccharide species in HS3is mirrored by a decrease in that species in the material not retainedby the BMP2 affinity column.

Statistical analysis was completed on the disaccharide composition asdetermined by the CE analysis of enzymatic digests (FIG. 77). The HS3preparations are significantly different from the starting material andthe material not retained by the BMP2 column for five of the eightcomponent disaccharides. The non-retained material is more like thestarting material differing significantly only in the proportion ofΔUA,2S-GlcNS,6S and ΔUA-GlcNS,6S, and this is consistent with thesepopulations being significantly enhanced in HS3 during the affinitychromatographic process.

In FIG. 78 it can be seen that there is no difference between the threeHS3 preparations (bulk material, initial run and final run) at the 95%confidence level. This is significant as it indicated that the column isoperating to selectively remove the same distribution of Celsus HS asHS3 throughout the affinity column process.

Example 11 Dot Blot Assay

To determine GAG-binding to BMP-2, we added 0.5 μg of BMP-2 in 200 μLPBS under vacuum onto a nitrocellulose membrane. The blot was blockedwith 5 percent BSA in PBS, prior to incubating each BMP2 spot with 1 μgof biotinylated GAG in 200 μL PBS. The bound biotinylated GAG wasdetected as described using HRP-conjugated streptavidin (BD Pharmingen).

We then compared the capacity of HS+ve to bind preferentially to BMP-2with that of the other HS compounds, using both GAG binding plates anddot blot assays (FIG. 80 a,b). The HS−ve showed weaker binding, andunfractionated HSpm showed only moderate binding, as compared to HS+ve.When GAG was omitted, we detected little BMP-2 binding to the plate(FIG. 80 a). FIG. 81 depicts the effect of dose on this assay. Dot blotassays further confirmed that only HS+ve bound strongly to BMP-2 (FIG.80 b).

We next examined the propensity of HSpm, HS+ve, and HS−ve to bind FGF-2,VEGF, and PDGF, other heparin-binding mitogenic peptides that areassociated with precursor cell growth and development (FIG. 80 c-e).Again, HS+ve demonstrated relatively poor affinity for these factors(FIG. 80 d). In a more stringent variation of this assay, using surfaceplasmon resonance, we analyzed how avidly HSpm, HS+ve, and HS−ve bind tothe structurally related BMP-2, BMP-4, and BMP-7 (SPR; Biacore 3000).HS+ve bound to BMP-2 strongly, and in a dose-dependent manner, withrelatively little binding to BMP-4 and BMP-7 (FIG. 82 a-c). In a furtheriteration, we showed that BMP-2 bound to the endogenous HS present onthe surfaces of C2C12 cells (FIG. 83 a) and could be effectivelycompeted off with HS+ve, but not with HSpm or HS−ve (FIG. 83 b).

The BMP-2-binding HS+ve that we isolated from commercially availableporcine mucosal HS(HSpm) increased specific BMP-2-binding five to tentimes over its parent sugar, and displayed little affinity for othertissue repair factors known to be abundant in wound sites, such as FGF,PDGF, and VEGF. Previous studies have shown that heparin enhancesBMP-2-induced osteoblast differentiation in C2C12 myoblasts in vitro(Katagiri, T. et al. Bone morphogenetic protein-2 converts thedifferentiation pathway of C2C12 myoblasts into the osteoblast lineage.J Cell Biol 127, 1755-1766 (1994)). Our results demonstrated that HS+vecan also push these cells into the osteoblast lineage. Previous work byZhao et al (Zhao, B. et al. Heparin potentiates the in vivo ectopic boneformation induced by bone morphogenetic protein-2. J Biol Chem 281,23246-23253 (2006)) has demonstrated that heparin can prolong BMP-2half-life in culture media, and Takada et al (Takada, T. et al. Sulfatedpolysaccharides enhance the biological activities of bone morphogeneticproteins. J Biol Chem 278, 43229-43235 (2003)) have suggested thatheparin maintains BMP-2 levels in culture media by preventing itsaccumulation within the extracellular matrix secreted by cells.

Our results provide ancillary evidence for both of these findings, asHS+ve both stabilized BMP-2 to maintain its activity, and also displacedBMP-2 from the cell surface on which intracellular localization anddegradation of BMP-2 subsequently takes place28. The addition of HS+veenhanced BMP-2 activity primarily by increasing its bioavailability.

Example 12 Alkaline Phosphatase Activity Assay

Myoblast C2C12 cells were maintained in DMEM, 10 percent FCS and 100U/mL P/S at 37° C., 5 percent CO₂. For the BMP-2-induced alkalinephosphatase (ALP) assay, C2C12 cells were seeded at 2×10⁴ cells/cm² in24-well plates in maintenance media. After 24 hours, the culture mediawere replaced with treatment media (maintenance media with FCS reducedto 5 percent) in the presence or absence of 100 ng/mL BMP-2 and 0.03 or0.3 or 3 μg/mL heparin, HSpm, HS+ve, or HS−ve. We cultured the cells intreatment media for 3 days, and after 3 days induction, we determinedthe ALP activity. Briefly, the cell layer was washed once withphosphate-buffered saline and lysed in RIPA buffer in the presence ofprotease inhibitor cocktail (Calbiochem) scraped with a rubber policemanand collected in 1.5 ml Eppendorf tubes. All steps were performed at 4°C. The protein content was determined using a BCA protein assay kit(Pierce Chemical Co.). ALP activity was measured by mixing 7 μg ofprotein with p-nitrophenylphosphate (Zymed). Enzyme activity wasmeasured as a change in absorbance at 405 nm due to the production ofp-nitrophenol per μg protein. The activity was normalized to treatmentcontaining BMP-2 alone and expressed as relative ALP activity.

ALP Staining

Cells were seeded in duplicate at a density of 20,000 cells/cm² in24-well plates in complete medium (10 percent FCS) and left to adherefor 24 hours. The following day, the cells were re-fed with the 5percent FCS medium, with or without BMP-2, together with one of threedifferent concentrations of HS (0.3 μg/ml, 3 ug/ml, or 30 μg/ml) andgrown thereafter for 3 days. After 3 days, the cells were fixed andstained for ALP using Sigma-Aldrich ALP staining kit.

Mineralization Assay

Myoblast C2C12 cells were seeded at 5×10³ cells/cm² in 24-well plates inmaintenance media. After 24 hours, we replaced the media with osteogenicmedia (DMEM, 5 percent FCS, 50 mg/ml ascorbic acid, and 10 mMb-glycerophosphate) in the presence/absence of 100 ng/mL BMP-2 and 5mg/mL GAG. We changed the media every 2 days. After 14 days, the celllayer was washed with PBS, fixed with 4 percent paraformaldehyde, andstained for 10 minutes in 0.1 percent alizarin red solution.

Anticoagulation Assay

GAGs were assessed for their effect on antithrombin III activity. Theassay was performed using the COATEST Heparin kit (Chromogenix)according to the manufacturer's specification. Values were representedas the relative inhibition of Factor Xa activity when compared totreatment group containing no GAG.

In Vitro BMP-2 Stability and Activity Assay

BMP-2 at 100 ng/mL was incubated alone or in the presence of 5 μg/mL ofGAG in treatment media at 37° C. and 5 percent CO². The media wascollected at the nominated time and stored at −80° C. prior to BMP-2quantitation. The amount of BMP-2 present in the media was assayed usinga BMP-2 Quantikine ELISA kit (R&D Systems) according to themanufacturer's specification.

BMP-2 Activity

C2C12 cells were cultured in 24-well plates, as described earlier. BMP-2in treatment media was prepared as described in the BMP-2 stabilityassay, for an indicated amount of time. Prior to adding the mediacontaining BMP-2, the cell layer was pre-incubated with fresh treatmentmedia for 24 hours. We then subjected the cells to BMP-2 for 15 minutesand lysed the cell layer in Laemmli buffer, which was in turn resolvedin a 4-12 percent SDS-PAGE gel, and immunoblotted with antibodiesagainst Smad 1/5/8 and phosphorylated Smad 1/5/8.

HS+ve Enhances BMP-2-Induced ALP Activity

We compared the relative abilities of HSpm, HS+ve, and HS−ve tostrengthen BMP-2 induction of ALP activity, which serves as a marker ofosteoblast differentiation in C2C12 cells. Alone, HSpm, HS+ve, and HS−vehad no effect; however, when cells were treated with 100 ng/ml of BMP-2in combination with HS+ve, the induction of ALP activity was greatlyenhanced (FIG. 85 a). At 5 μg/mL, both HSpm and HS+ve significantlyenhanced BMP-2-induced ALP activity, with HS+ve significantly superiorcompared to HSpm (FIG. 85 a). Phenotypic staining for ALP confirmed thatcultures treated with HS+ve at 5 μg/ml had greater densities ofALP-positive cells, unlike those treated with HSpm and HS−ve (FIG. 85b).

Once we determined that HS+ve enhanced BMP-2 activity after 3 days, wenext identified its long-term effects on matrix mineralization, asassessed by Alizarin Red staining after 14 days of culture. Strikingly,long-term HS+ve supplementation greatly enhanced BMP-2-stimulated matrixmineralization, yet neither HSpm nor HS−ve had any effect (FIG. 86).

HS+ve Reduces Noggin Activity but has Little Anticoagulant Activity

The antagonism provided by noggin, a secreted BMP-2 inhibitor, regulatesBMP-2's extracellular activity. To determine the effect of HS on noggin,we assayed for ALP activity in C2C12 cells after exposure to BMP-2, HS,and noggin combinations. Noggin at 30 and 100 ng/ml significantlyinhibited BMP-2-induced ALP activity by 14 and 95 percent respectively(FIG. 85 c). Noggin's inhibitory effect at 30 and 100 ng/ml weresignificantly reduced to 2 and 43 percent inhibition respectively in thepresence of HS+ve and to 4 and 62 percent respectively in the presenceof HSpm.

However, the inhibition of BMP-2-induced ALP activity in the presence ofHS+ve and

HSpm was still significant at 100 ng/ml of noggin. HS−ve alsosignificantly reduced noggin's inhibitory effect at 100 ng/mL but stillmaintained 75 percent inhibition. Based on analysis of main effects intwo-way ANOVA, we found that HS+ve was the most effective compared toother HS variants in reducing noggin's inhibitory activity, while HS−vewas the least effective. In order to confirm that HS+ve possessed littleanticoagulant activity, we compared its ability to activateanti-thrombin III to that of heparin. We found that heparin activatedanti-thrombin III in a dose-dependent manner, thus inhibiting Factor Xa,but HS+ve could not produce the same effect (FIG. 87).

The presence of antagonists also influences BMP-2's bioavailability. Forexample, heparin has been shown to reduce the antagonistic effect ofnoggin (Zhao, B. et al. Heparin potentiates the in vivo ectopic boneformation induced by bone morphogenetic protein-2. J Biol Chem 281,23246-23253 (2006), Paine-Saunders, S., Viviano, B. L., Economides, A.N. & Saunders, S. Heparan sulphate proteoglycans retain Noggin at thecell surface: a potential mechanism for shaping bone morphogeneticprotein gradients. J Biol Chem 277, 2089-2096 (2002). In this study, weshowed that HS+ve induced activation by also preventing the interactionbetween BMP-2 and noggin. Neither HSpm nor HS−ve enhanced the stabilityof BMP-2, and neither prevented BMP-2/noggin interactions. Based on ourin vitro results, we posit that without HS, BMP-2 degrades quickly andbecomes particularly susceptible to its antagonist but that in thepresence of an appropriate HS, BMP2 becomes more bioavailable.

Example 13 Luciferase Reporter Assay

To determine the transcription activity of BMP signaling, 0.8 μg Id1promoter luciferase reporter, which contains a BMP-responsive element31,and 8 ng Renilla-LUC vector (Promega, USA) were transfected into4×10⁴C2C12 cells with Lipofectamine 2000 (Invitrogen Life Technologies,USA) for 6 hours, prior to treatment with BMP-2, HS+ve, or heparin foranother 72 hours. Luciferase activity was then determined using theDual-Luciferase assay kit (Promega, USA) according to the manufacturer'sinstructions. Briefly, cells were lysed in the PLB buffer supplied andthe activities of firefly (produced by id1-LUG) and Renilla luciteraseswere measured sequentially in each sample. The Id1 luciferase activitieswere normalized by Renilla-LUC activity. Each condition was tested intriplicate, and the experiment was repeated in triplicate for threeindependent experiments.

6×OSE Luciferase Reporter Assay

To determine RUNX2 transcriptional activity, cells were transfected with0.4 μg of 6×OSE-LUC vector32 and 4 ng of Renilla-LUC reporter vector.Six hours later, cells were treated as indicated for 24 hours and theluciferase activity examined with the Dual-Luciferase assay kit(Promega), according to the manufacturer's instructions. The activitiesof firefly and Renilla luciferases were measured sequentially in eachsample and the firefly luciferase activity normalized to the Renillaluciferease activity.

Example 14 Pro-Inflammatory Responses

Mouse macrophages (RAW264.7, ATCC, USA) were used to evaluate thepro-inflammatory response to HS/Col composites. Cells were cultured inATCC-formulated DMEM supplemented with 10 percent FCS, 2 mMpenicillin/streptomycin and L-glutamine and incubated in 5 percent CO₂at 37° C.

Media was changed every other day and RAW264.7 cells (passages 6-8) wereused for proinflammatory experiments. Short-term inflammatory responsesto the HS/Col composites were evaluated over 24 hours and assessed bymeasuring endogenous levels of tumor necrosis factor alpha (TNF-α) inthe culture media (Luong-Van, E. et al. Controlled release of heparinfrom poly(epsilon-caprolactone) electrospun fibers. Biomaterials 27,2042-2050 (2006)). Cells were seeded (3×10⁵ cells/cm²) in 48-well platesand cultured for 24 hours in the presence of HS-conditioned media ormedia containing exogenous HS (5 μg). The amount of TNF-α secreted intothe media in response to the treatment media was quantified using TNF-αELISA kit (BD Bioscience, USA) per the manufacturer's instructions.Cells stimulated with 10 ng/mL lipopolysaccharide (LPS, Sigma, USA) wereused as a positive control, while cells grown in basal media withoutstimulation provided measurement of background TNF-α levels.

Example 15 HS/BMP2 Stimulates Robust Bone Regeneration in Micro-PigMandibular Defects

When the alveolar ride bone is compromised due to periodontal disease ortrauma, rehabilitation with dental implant is difficult. Current ‘goldstandard’ treatment requires the transplantation of an autograft takenfrom the patient's donor site, usually the 3rd molar region to rebuildsufficient bone for anchorage of dental implant. This is associated withmultiple surgeries and donor site morbidity. Thus, the challenge is toprovide effective graft alternatives to ensure sufficient bone volume1available for dental implant therapy that allows predictable and dentalreconstruction outcomes with minimal clinical complications.

10 male micro-pigs were operated on as previously described (Yeo, A. et.al., Lateral ridge augmentation using a PCL-TCP scaffold in a clinicallyrelevant but challenging micropig model. Clin Oral Implants Res 2011Dec. 6. doi: 10.1111/j.1600-0501.2011.02366.x.). As such, 2 criticalsized alveolar bone defects (12×8×5 mm) were created per side ofmicro-pig mandible, followed by a 3 month healing period.Polycaprolactone-20% tricalcium phosphate (PCL-TCP) scaffolds with aunique 3D micro-architecture3 purchased from Osteopore International,Singapore. Tisseel Sealant purchased from Baxter Immuno, USA.Recombinant Human Bone morphogenetic protein-2 (BMP-2) purchased fromR&D Systems, USA. BioGide® collagen membrane purchased from GeistlichPharma AG, Switzerland. Resin histology4, X-ray and p-CT5 analyses wereperformed. For implantation of scaffolds, 3 groups were formed(n=3/group): (1) PCL-TCP only (control); (2) (1)+10 μg BMP-2; (3) (1)+30μg HS3. Animals were sacrificed at 3 and 6 months post treatment.Analysis was by 2d X-ray, 3D p-CT scan and resin histology.

2 treatments were implanted on each side of a mandible and allowed torecover for up to 6 months (see FIG. 90). At both 3 and 6 monthspost-implantation, p-CT images showed more new bone formation in boththe 10 μg BMP-2 and 30 μg HS3 treatments than scaffold alone (FIGS. 91and 92). Notably, 30 μg HS3 was able to form as much alveolar bone as 10μg BMP-2. Results are presented as mean±standard error of mean andsignificance is taken as p<0.05.

This pilot preclinical study in micro-pigs clearly demonstrated that thePCL-TCP scaffold, when combined with a stand-alone treatment of 30 μgHS3 increased alveolar ridge bone volume significantly. The newly formedbone will assist in the anchorage of prosthetic implants and eliminatethe need for additional bone graft surgery.

Example 16 Healing Rat Calvarial Critical-Sized Bone Defects with BoneMorphogenetic Protein2-Binding Heparan Sulfate

Over 10% of fractures go on to either delayed union or non-union (1.Garrison K R et al. Clinical effectiveness and cost-effectiveness ofbone morphogenetic proteins in the non-healing of fractures and spinalfusion: a systematic review. Health Technol Assess 2007; 11.) As such, abioactive agent to stimulate bone formation for the treatment offracture healing and to replace substantial bone loss seen inosteoporosis patients is needed.

The use of BMP-2 is currently hindered by cost and rapid clearance rate(Ruhe P Q et al. In vivo release of rhBMP-2 loaded porous calciumphosphate cement pretreated with albumin. J Mater Sci Mater Med 2006;17:919e27.). The FDA issued a warning regarding the off-label use ofrhBMP-2 on Jul. 7, 2008, reporting at least 38 cases of complicationsassociated with using BMP-2 for cervical fusion.

The extracellular glycosaminoglycan sugar, heparan sulfate (HS), bindsto a variety of soluble proteins involved in controlling cell phenotype,such BMPs, so increasing their binding to high affinity receptors (Rai Bet al. Heparan sulfate-based treatments for regenerative medicine. Crit.Rev Euk Gene Exp 2011; 21:1-12. 4).

The HS chains of proteoglycans not only protect the growth factor fromnormal proteolytic degradation, but also enhance and stabiliseligand/receptor interactions on cell surfaces (Bramono D S et al. Bonemarrow-derived heparan sulfate potentiates the osteogenic activity ofbone morphogenetic protein-2. Bone 2012; 50:954-964).

As described herein, we designed a simple purification method for aBMP-2-binding-HS fraction (called HS/BMP2 or HS3) from crude HS mixture,based on a unique heparin-binding domain for BMP-2 and established thefollowing in vitro:

-   -   HS/BMP2 binds specifically to BMP-2    -   HS/BMP2 enhances BMP-2 induced ostoegenesis in C2C12 cells    -   HS/BMP2 prolongs BMP-2's half-life    -   HS/BMP2 sustains BMP-2's bioactivity    -   HS/BMP2 reduces activity between BMP-2 and antagonist noggin.

We hypothesised that a single bolus of HS3 alone will prove to be justas effective as BMP-2 for the healing of critical sized calvarialdefects in rats.

Experimental Groups

1. Fibrin Sealant (Baxter)/PCL-TCP scaffolds (Ctrl, OsteoporeInternational)2. (1)+5 μg rhBMP-2 (R & D Systems)3. (1)+3 μg HS3+ve (BMP-2 binding fraction)4. (1)+30 μg HS3+ve (BMP-2 binding fraction)5. (1)+3 μg HS3PM (Crude HS mixture)6. (1)+30 μg HS3PM (Crude HS mixture)7. (1)+3 μg HS3-ve (non-binding fraction)8. (1)+30 μg HS3-ve (non-binding fraction)

Methods of Analysis New Bone Assessment At 12 Weeks:

-   -   Standard 2D X-rays (MUX-100) & 3D μ-CT (Skyscan 1076), N=6/group    -   Paraffin Histology (Hematoxylin & Eosin, Modified Trichrome        stains)    -   Immunohistochemistry (Collagen Type I & Osteocalcin) 5-6

Retention of BMP-2 and HS3+ve:

-   -   BMP-2 & HS3+ve was labeled with XenoLight CF₆₈₀ and XenoLight        IF750 respectively (N=3/group).    -   Imaged using IVIS Spectrum (Caliper Life Sciences) and analyzed        with Living Image 3.1.

Results

Gross morphology was followed by sequential standard 2D x-rays and 3Dp-CT scanning. New bone filling up the defect was evident in the 5 μgBMP-2 and 30 μg HS3+ve-treated calvarial defects (FIG. 93A).

Areas of new bone were observed throughout the defects treated withBMP-2 and HS3+ve. The scaffold was still evident at 12 weeks asindicated by their porous morphology. Controls demonstrated only a smallamount of new bone, which was confined to the edges of the defect (FIG.93B).

There was clear evidence of accelerated new bone formation in defectstreated with 30 μg of HS3+ve, at an extent comparable to BMP-2-treateddefects. In contrast, HS^(PM) and HS3-ve treated defects showed onlymodest amounts of newly regenerated bone, similar to control (FIG. 93C).

FIG. 93D illustrates a representative rat per treatment over a period of28 days. The results showed that both BMP-2 and HS3 were retained invivo at a similar extent, to a maximum of 21 days. Interestingly, HS3distribution was consistent in all 3 rats and there was no detectablesignal peripheral to the delivery site. This was in contrast to BMP-2,where distribution was dissimilar in the 3 rats and a signal wasdetected peripheral to the defect site as early as 2 days afterimplantation, which may impose health risks.

In conclusion, we found that a single bolus of HS3+ve alone proved to bejust as effective as BMP-2 for the healing of critical-sized calvarialdefects in rats. BMP-2 and HS3+ve were detected in the rats for up to 21days. Notably, HS3+ve remained localised to the defect site, unlikeBMP-2 that spread away as early as 2 days. Complete healing of thedefects was not achieved at 12 weeks, hence a longer-time point isrecommended as future work.

Example 17 Heparan Glycosaminoglycans Lower the Therapeutic Dose ofBMP-2

Autologous bone graft remains the gold standard for the treatment oforthopaedic trauma, however donor-site morbidity and insufficient graftmaterial limit its use (Arrington, E. D., Smith, W. J., Chambers, H. G.,Bucknell, A. L. & Davino, N. A. Complications of iliac crest bone graftharvesting. Clin Orthop Relat Res, 300-309 (1996). Banwart, J. C.,Asher, M. A. & Hassanein, R. S. Iliac crest bone graft harvest donorsite morbidity. A statistical evaluation. Spine (Phila Pa 1976) 20,1055-1060 (1995)). Although bone graft substitutes containing BMP-2 areefficacious, supra-physiological doses are used clinically due to itsinherent instability (Bishop, G. B. & Einhorn, T. A. Current and futureclinical applications of bone morphogenetic proteins in orthopaedictrauma surgery. Int Orthop 31, 721-727 (2007)). As such, BMP-2 treatmentis both costly and associated with adverse dose-related osteopathologies(Carragee, E. J., Hurwitz, E. L. & Weiner, B. K. A critical review ofrecombinant human bone morphogenetic protein-2 trials in spinal surgery:emerging safety concerns and lessons learned. Spine J 11, 471-491(2011)). An important therapeutic aim is therefore to lower theefficacious dose of BMP-2. Heparan sulfate (HS), a glycosaminoglycanstructural analog of heparin that has a greater specificity of sulfationmay provide greater selectivity in protein interactions (David, G. &Bernfield, M. The emerging roles of cell surface heparan sulfateproteoglycans. Matrix Biol 17, 461-463 (1998), Turnbull, J., Powell, A.& Guimond, S. Heparan sulfate: decoding a dynamic multifunctional cellregulator. Trends Cell Biol 11, 75-82 (2001), Kuo, W. J., Digman, M. A.& Lander, A. D. Heparan sulfate acts as a bone morphogenetic proteincoreceptor by facilitating ligand-induced receptorhetero-oligomerization. Mol Biol Cell 21, 4028-4041 (2010)). This uniquecapability can be exploited to improve the therapeutic utility of BMP-2.

Our aims were to evaluate the use of HS tuned to preferentially bind toand activate BMP-2 (called HS/NMP2 or HS3) on its ability to enhanceBMP-2-mediated osteogenic activity in vitro and to investigate the boneinducing efficacy of HS3 delivered by a type I bovine collagen sponge(Helistat®), in critical-sized ulna defects in rabbits.

Experimental Design

In vitro: To investigate the capacity of HS3 for BMP-2 compared to otherheparin binding growth factors using GAG binding plates. To evaluate theability of HS3 to augment BMP-2 induction of ALP activity, a marker ofosteoblast differentiation, in C2C12 cels. To confirm that HS3 canstabilise BMP-2 activity, Smad phosphorylation was monitored in C2C12cells at 24, 48 and 72 h.

In vivo: Treatments (Collagen sponge only or with 10 μg BMP-2 or 30 μgHS3) were surgically implanted into critical-sized ulna defects inrabbits and bone regeneration was determined at 8 weekspost-implantation by 2D X-rays, 3D p-CT analysis, mechanical testing andparaffin histology.

Results

HS3 bound preferentially to BMP-2 and demonstrated relatively pooraffinity for other heparin binding growth factors (FIG. 94A). HS3 had noeffect alone on ALP activity in C2C12 cells; however, when cells weretreated with 100 ng/ml of BMP-2 in combination with HS3, the inductionof ALP activity was greatly enhanced over that seen in BMP-2 alone (FIG.94B). We observed BMP-2-induced p-Smad 1/5/8 activity at comparableintensities, with or without HS3 at 24 hours. However, after 48 hours,the presence of HS3 maintained the p-Smad 1/5/8 signaling (FIG. 94C).

Critical-sized ulna defects in rabbits were treated with collagen spongealone (ctrl), 10 μg BMP-2 or 30 μg HS3 for 8 weeks. X-rays showedcomplete bridging of defects in both HS3 and BMP-2 treatments (FIG. 95).3D p-CT images confirmed the presence of mineralized bone and remodelingof the cortical shell (FIG. 95). Hematoxylin & eosin stainingillustrated the infiltration of new bone from the radial interface andhost bone at the osteotomized ends (FIG. 95). Expression of both early(collagen type I) and late (osteocalcin) osteogenic markers were evidentin the HS3- and BMP-2 treated-defects (FIG. 95).

The extent of mineralization was measured by μ-CT at 8 weeks. HS3treatment generated significantly higher bone volume than control, withBMP-2 having a similar effect (FIG. 96A). Torsional testing of treateddefects was conducted at 8 weeks. The torsional stiffness and maximumtorque were restored for both HS3 and BMP-2-treated defects, on par withintact bone (FIG. 96B).

The data obtained indicates that HS3 interacts with BMP-2 and sustainsits bioactivity in vitro. This translates into augmented bone formationin vivo. Hence, this study provides a means to explore the therapeuticuse of HS3 as a bone healing adjuvant.

1. Isolated or substantially purified heparan sulphate HS/BMP2, whereinfollowing digestion with heparin lyases I, II and III and thensubjecting the resulting disaccharide fragments to capillaryelectrophoresis analysis the heparan sulphate HS/BMP2 has a disaccharidecomposition comprising: Normalised weight Disaccharide percentageΔHexUA,2SGlcNS,6S 14.8 ± 3.0 ΔHexUA,2S-GlcNS  4.9 ± 2.0 ΔHexUA-GlcNS,6S11.1 ± 3.0 ΔHexUA,2SGlcNAc,6S  4.8 ± 2.0 ΔHexUA-GlcNS 22.2 ± 3.0ΔHexUA,2S-GlcNAc  1.1 ± 0.5 ΔHexUA-GlcNAc,6S 10.1 ± 3.0 ΔHexUA-GlcNAc31.1 ± 3.0


2. Isolated or substantially purified heparan sulphate HS/BMP2 accordingto claim 1 wherein following digestion with heparin lyases I, II and IIIand then subjecting the resulting disaccharide fragments to capillaryelectrophoresis analysis the heparan sulphate HS/BMP2 has a disaccharidecomposition comprising: Normalised weight Disaccharide percentageΔHexUA,2SGlcNS,6S 14.8 ± 1.0 ΔHexUA,2S-GlcNS  4.9 ± 0.4 ΔHexUA-GlcNS,6S11.1 ± 1.0 ΔHexUA,2SGlcNAc,6S  4.8 ± 0.6 ΔHexUA-GlcNS 22.2 ± 3.0ΔHexUA,2S-GlcNAc  1.1 ± 0.4 ΔHexUA-GlcNAc,6S 10.1 ± 1.0 ΔHexUA-GlcNAc31.1 ± 1.6


3. HS/BMP2 according to claim 1 wherein the HS/BMP2 is capable ofbinding SEQ ID NO:1 or
 6. 4. A pharmaceutical composition or medicamentcomprising HS/BMP2 according to claim
 1. 5. The pharmaceuticalcomposition or medicament of claim 4 further comprising BMP2 protein. 6.A biocompatible implant or prosthesis comprising a biomaterial andisolated or substantially purified HS/BMP2 according to claim
 1. 7. Theimplant or prosthesis of claim 6 wherein the implant or prosthesis iscoated with said HS/BMP2.
 8. The implant or prosthesis of claim 6wherein the implant or prosthesis is impregnated with said HS/BMP2.
 9. Amethod of treating a bone fracture in a patient, the method comprisingadministration of a therapeutically effective amount of HS/BMP2according to claim 1 to the patient, thereby treating the bone fracture.10. The method of claim 9 wherein the method comprises administeringsaid HS/BMP2 to the tissue surrounding the fracture.
 11. The method ofclaim 9 wherein the HS/BMP2 is formulated as a pharmaceuticalcomposition or medicament comprising HS/BMP2 and a pharmaceuticallyacceptable carrier, adjuvant or diluent.
 12. The method of claim 9wherein the method further comprises administering BMP2 protein to thepatient.